Engineered CAS9 systems for eukaryotic genome modification

ABSTRACT

Engineered Cas9 systems that utilize alternate protospacer adjacent motifs for target DNA binding, nucleic acids encoding the engineered Cas9 systems, and methods of using the engineered Cas9 systems for modifying target chromosomal sequences in eukaryotic cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/631,304, filed Feb. 15, 2018, and U.S. Provisional ApplicationSer. No. 62/720,525, filed Aug. 21, 2018, the disclosure of each ofwhich is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 12, 2019, isnamed P18_023US_SL.txt and is 367,427 bytes in size.

FIELD

The present disclosure relates to engineered Cas9 systems, nucleic acidsencoding said systems, and methods of using said systems for genomemodification.

BACKGROUND

The recent development of the bacterial class 2 Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated(Cas) CRISPR/Cas systems as genome editing tools has providedunprecedented ease and simplicity to engineer site-specificendonucleases for eukaryotic genome modification. However, because eachCRISPR/Cas system requires a specific protospacer adjacent motif (PAM)for target DNA binding, each system is limited to certain genomic sites.Although the currently most widespread adopted Streptococcus pyogenesCas9 (SpyCas9) uses a frequently occurring PAM (5′-NGG-3′) fortargeting, it is still excluded from many genomic sites lacking such amotif, since eukaryotic genomes, especially those of mammals and plants,are highly complex and heterogeneous in DNA sequence. Moreover,precision gene editing using homology-directed repair (HDR) or baseeditors such as dCas9/cytidine deaminase and dCas9/adenosine deaminaseoften requires a precise DNA binding position, even at the single basepair resolution, to achieve an optimal editing outcome. Therefore, thereis a need to develop new CRISPR/Cas systems that use novel PAMs fortargeting to increase genome coverage density.

SUMMARY

Among the various aspects of the present disclosure include engineeredCas9 systems comprising engineered Cas9 proteins and engineered guideRNAs, wherein each engineered guide RNA is designed to complex with anengineered Cas9 protein and the engineered guide RNA comprises a 5′guide sequence designed to hybridize with a target sequence in adouble-stranded sequence, wherein the target sequence is 5′ to aprotospacer adjacent motif (PAM) and the PAM has a sequence as listed inTable A.

Another aspect of the present disclosure encompasses a plurality ofnucleic acids encoding said engineered Cas9 systems and at least onevector comprising the plurality of said nucleic acids.

A further aspect includes eukaryotic cells comprising at least oneengineered Cas9 system and/or at least one nucleic acid encoding saidengineered Cas9 system.

Still another aspect of the present disclosure encompasses methods formodifying chromosomal sequences in eukaryotic cells. The methodscomprise introducing into the eukaryotic cell at least one engineeredCas9 system comprising an engineered Cas9 protein and an engineeredguide RNA and/or at least one nucleic acid encoding said engineered Cas9system and, optionally, at least one donor polynucleotide, wherein theat least one engineered guide RNA guides the at least one engineeredCas9 protein to the target site in the chromosomal sequence such thatmodification of the chromosomal sequence occurs.

Other aspects and features of the disclosure are detailed bellow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the WebLogo analysis of protospacer adjacent motifs (PAM)required for in vitro target DNA cleavage by Cas9 orthologs. Numbers onthe horizontal axis indicate the position of the nucleotide in the PAMsequence.

FIG. 2A presents the cleavage efficiency (as the percent of indels) ofMcaCas9, McaCas9-HN1HB1 fusion (i.e., HMGN1 at the amino terminus andHMGB1 box A at the carboxyl terminus), and McaCas9-HN1H1G fusion (i.e.,HMGN1 at the amino terminus and histone H1 central globular motif at thecarboxyl terminus). The target site of each locus is presented in Table6. Error bars show mean±SD (n=3 biological replicates).

FIG. 2B presents the cleavage efficiency (as the percent of indels) ofPexCas9, PexCas9-HN1HB1 fusion (i.e., HMGN1 at the amino terminus andHMGB1 box A at the carboxyl terminus), and PexCas9-HN1H1G fusion (i.e.,HMGN1 at the amino terminus and histone H1 central globular motif at thecarboxyl terminus). The target site of each locus is presented in Table6. Error bars show mean±SD (n=3 biological replicates).

FIG. 2C presents the cleavage efficiency (as the percent of indels) ofBsmCas9, BsmCas9-HN1HB1 fusion (i.e., HMGN1 at the amino terminus andHMGB1 box A at the carboxyl terminus), and BsmCas9-HN1H1G fusion (i.e.,HMGN1 at the amino terminus and histone H1 central globular motif at thecarboxyl terminus). The target site of each locus is presented in Table6. Error bars show mean±SD (n=3 biological replicates).

FIG. 2D presents the cleavage efficiency (as the percent of indels) ofLrhCas9, LrhCas9-HN1HB1 fusion (i.e., HMGN1 at the amino terminus andHMGB1 box A at the carboxyl terminus), and LrhCas9-HN1H1G fusion (i.e.,HMGN1 at the amino terminus and histone H1 central globular motif at thecarboxyl terminus). The target site of each locus is presented in Table6. Error bars show mean±SD (n=3 biological replicates).

FIG. 3 shows off-target activities (as the percent of indels) of controlCas9 and Cas9-CMM fusion nucleases. Error bars show mean±SD (n=3biological replicates).

DETAILED DESCRIPTION

The present disclosure provides orthologous Cas9 systems that usealternate PAMs for target DNA binding, thereby increasing genomecoverage density. For example, some of these alternate PAMs comprise Aand/or T residues, and other alternate PAMS are GC-rich. As such, theengineered Cas9 systems that utilize these alternate PAMs enabletargeted genome editing or genome modification of previouslyinaccessible genomic loci.

(I) Engineered Cas9 Systems

One aspect of the present disclosure provides engineered Cas9 systemscomprising engineered Cas9 proteins and engineered guide RNAs, whereineach engineered guide RNA is designed to complex with a specificengineered Cas9 protein. Each engineered guide RNA comprises a 5′ guidesequence designed to hybridize with a target sequence in adouble-stranded sequence, wherein the target sequence is 5′ to aprotospacer adjacent motif (PAM) and the PAM has a sequence as listed inTable A. These engineered Cas9 systems do not occur naturally.

(a) Engineered Cas9 Proteins

The engineered Cas9 protein comprises at least one amino acidsubstitution, insertion, or deletion relative to its wild-typecounterpart. Cas9 protein is the single effector protein in type IICRISPR systems, which are present in various bacteria. The engineeredCas9 protein disclosed herein can be from Acaryochloris sp.,Acetohalobium sp., Acidaminococcus sp., Acidithiobacillus sp.,Acidothermus sp., Akkermansia sp., Alicyclobacillus sp., Allochromatiumsp., Ammonifex sp., Anabaena sp., Arthrospira sp., Bacillus sp.,Bifidobacterium sp., Burkholderiales sp., Caldicelulosiruptor sp.,Campylobacter sp., Candidatus sp., Clostridium sp., Corynebacterium sp.,Crocosphaera sp., Cyanothece sp., Exiguobacterium sp., Finegoldia sp.,Francisella sp., Ktedonobacter sp., Lachnospiraceae sp., Lactobacillussp., Lyngbya sp., Marinobacter sp., Methanohalobium sp., Microscillasp., Microcoleus sp., Microcystis sp., Mycoplasma sp., Natranaerobiussp., Neisseria sp., Nitratifractor sp., Nitrosococcus sp., Nocardiopsissp., Nodularia sp., Nostoc sp., Oenococcus sp., Oscillatoria sp.,Parasutterella sp., Pelotomaculum sp., Petrotoga sp., Polaromonas sp.,Prevotella sp., Pseudoalteromonas sp., Ralstonia sp., Staphylococcussp., Streptococcus sp., Streptomyces sp., Streptosporangium sp.,Synechococcus sp., Thermosipho sp., Verrucomicrobia sp., and Wolinellasp.

In certain embodiments, the engineered Cas9 protein disclosed herein isfrom Acidothermus sp., Akkermansia sp., Alicyclobacillus sp., Bacillussp., Bifidobacterium sp., Burkholderiales sp., Corynebacterium sp.,Lactobacillus sp., Mycoplasma sp., Nitratifractor sp., Oenococcus sp.,Parasutterella sp., Ralstonia sp., or Wolinella sp.

In specific embodiments, the engineered Cas9 protein disclosed herein isfrom Acidothermus cellulolyticus (Ace), Akkermansia glycaniphila (Agl),Akkermansia muciniphila (Amu), Alicyclobacillus hesperidum (Ahe),Bacillus smithii (Bsm), Bifidobacterium bombi (Bbo), Corynebacteriumdiphtheria (Cdi), Lactobacillus rhamnosus (Lrh), Mycoplasma canis (Mca),Mycoplasma gallisepticum (Mga), Nitratifractor salsuginis (Nsa),Oenococcus kitaharae (Oki), Parasutterella excrementihominis (Pex),Ralstonia syzygii (Rsy), or Wolinella succinogenes (Wsu).

Wild-type Cas9 proteins comprise two nuclease domains, i.e., RuvC andHNH domains, each of which cleaves one strand of a double-strandedsequence. Cas9 proteins also comprise REC domains that interact with theguide RNA (e.g., REC1, REC2) or the RNA/DNA heteroduplex (e.g., REC3),and a domain that interacts with the protospacer-adjacent motif (PAM)(i.e., PAM-interacting domain).

The Cas9 protein can be engineered to comprise one or more modifications(i.e., a substitution of at least one amino acid, a deletion of at leastone amino acid, an insertion of at least one amino acid) such that theCas9 protein has altered activity, specificity, and/or stability.

For example, Cas9 protein can be engineered by one or more mutationsand/or deletions to inactivate one or both of the nuclease domains.Inactivation of one nuclease domain generates a Cas9 protein thatcleaves one strand of a double-stranded sequence (i.e., a Cas9 nickase).The RuvC domain can be inactivated by mutations such as D10A, D8A,E762A, and/or D986A, and the HNH domain can be inactivated by mutationssuch as H840A, H559A, N854A, N856A, and/or N863A (with reference to thenumbering system of Streptococcus pyogenes Cas9, SpyCas9). Inactivationof both nuclease domains generates a Cas9 protein having no cleavageactivity (i.e., a catalytically inactive or dead Cas9).

The Cas9 protein can also be engineered by one or more amino acidsubstitutions, deletions, and/or insertions to have improved targetingspecificity, improved fidelity, altered PAM specificity, decreasedoff-target effects, and/or increased stability. Non-limiting examples ofone or more mutations that improve targeting specificity, improvefidelity, and/or decrease off-target effects include N497A, R661A,Q695A, K810A, K848A, K855A, Q926A, K1003A, R1060A, and/or D1135E (withreference to the numbering system of SpyCas9).

(i) Heterologous Domains

The Cas9 protein can be engineered to comprise at least one heterologousdomain, i.e., Cas9 is fused to one or more heterologous domains. Insituations in which two or more heterologous domains are fused withCas9, the two or more heterologous domains can be the same or they canbe different. The one or more heterologous domains can be fused to the Nterminal end, the C terminal end, an internal location, or combinationthereof. The fusion can be direct via a chemical bond, or the linkagecan be indirect via one or more linkers. In various embodiments, theheterologous domain can be a nuclear localization signal, acell-penetrating domain, a marker domain, a chromatin disrupting domain,an epigenetic modification domain (e.g., a cytidine deaminase domain, ahistone acetyltransferase domain, and the like), a transcriptionalregulation domain, an RNA aptamer binding domain, or a non-Cas9 nucleasedomain.

In some embodiments the one or more heterologous domains can be anuclear localization signal (NLS). Non-limiting examples of nuclearlocalization signals include PKKKRKV (SEQ ID NO:78), PKKKRRV (SEQ IDNO:79), KRPAATKKAGQAKKKK (SEQ ID NO:80), YGRKKRRQRRR (SEQ ID NO:81),RKKRRQRRR (SEQ ID NO:82), PAAKRVKLD (SEQ ID NO:83), RQRRNELKRSP (SEQ IDNO:84), VSRKRPRP (SEQ ID NO:85), PPKKARED (SEQ ID NO:86), PQPKKKPL (SEQID NO:87), SALIKKKKKMAP (SEQ ID NO:88), PKQKKRK (SEQ ID NO:89),RKLKKKIKKL (SEQ ID NO:90), REKKKFLKRR (SEQ ID NO:91),KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:92), RKCLQAGMNLEARKTKK (SEQ ID NO:93),NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO:94), andRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:95).

In other embodiments, the one or more heterologous domains can be acell-penetrating domain. Examples of suitable cell-penetrating domainsinclude, without limit, GRKKRRQRRRPPQPKKKRKV (SEQ ID NO:96),PLSSIFSRIGDPPKKKRKV (SEQ ID NO:97), GALFLGWLGAAGSTMGAPKKKRKV (SEQ IDNO:98), GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO:99),KETWWETWWVTEWSQPKKKRKV (SEQ ID NO:100), YARAAARQARA (SEQ ID NO:101),THRLPRRRRRR (SEQ ID NO:102), GGRRARRRRRR (SEQ ID NO:103), RRQRRTSKLMKR(SEQ ID NO:104), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:105),KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:106), and RQIKIWFQNRRMKWKK(SEQ ID NO:107).

In alternate embodiments, the one or more heterologous domains can be amarker domain. Marker domains include fluorescent proteins andpurification or epitope tags. Suitable fluorescent proteins include,without limit, green fluorescent proteins (e.g., GFP, eGFP, GFP-2,tagGFP, turboGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP,AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP,Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins(e.g., BFP, EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire,T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet,AmCyanl, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate,mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2,DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry,mStrawberry, Jred), orange fluorescent proteins (e.g., mOrange, mKO,Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato), orcombinations thereof. The marker domain can comprise tandem repeats ofone or more fluorescent proteins (e.g., Suntag). Non-limiting examplesof suitable purification or epitope tags include 6×His (SEQ ID NO: 134),FLAG®, HA, GST, Myc, SAM, and the like. Non-limiting examples ofheterologous fusions which facilitate detection or enrichment of CRISPRcomplexes include streptavidin (Kipriyanov et al., Human Antibodies,1995, 6(3):93-101), avidin (Airenne et al., Biomolecular Engineering,1999, 16(1-4):87-92), monomeric forms of avidin (Laitinen et al.,Journal of Biological Chemistry, 2003, 278(6):4010-4014), peptide tagswhich facilitate biotinylation during recombinant production (Cull etal., Methods in Enzymology, 2000, 326:430-440).

In still other embodiments, the one or more heterologous domain can be achromatin modulating motif (CMM). Non-limiting examples of CMMs includenucleosome interacting peptides derived from high mobility group (HMG)proteins (e.g., HMGB1, HMGB2, HMGB3, HMGN1, HMGN2, HMGN3a, HMGN3b,HMGN4, and HMGN5 proteins), the central globular domain of histone H1variants (e.g., histone H1.0, H1.1, H1.2, H1.3, H1.4, H1.5, H1.6, H1.7,H1.8, H1.9, and H.1.10), or DNA binding domains of chromatin remodelingcomplexes (e.g., SWI/SNF (SWItch/Sucrose Non-Fermentable), ISWI(Imitation SWItch), CHD (Chromodomain-Helicase-DNA binding), Mi-2/NuRD(Nucleosome Remodeling and Deacetylase), INO80, SWR1, and RSC complexes.In other embodiments, CMMs also can be derived from topoisomerases,helicases, or viral proteins. The source of the CMM can and will vary.CMMs can be from humans, animals (i.e., vertebrates and invertebrates),plants, algae, or yeast. Non-limiting examples of specific CMMs arelisted in the table below. Persons of skill in the art can readilyidentify homologs in other species and/or the relevant fusion motiftherein.

Protein Accession No. Fusion Motif Human HMGN1 P05114 Full length HumanHMGN2 P05204 Full length Human HMGN3a Q15651 Full length Human HMGN3bQ15651-2 Full length Human HMGN4 O00479 Full length Human HMGN5 P82970Nucleosome binding motif Human HMGB1 P09429 Box A Human histone H1.0P07305 Globular motif Human histone H1.2 P16403 Globular motif HumanCHD1 O14646 DNA binding motif Yeast CHD1 P32657 DNA binding motif YeastISWI P38144 DNA binding motif Human TOP1 P11387 DNA binding motif Humanherpesvirus 8 J9QSF0 Nucleosome binding motif LANA Human CMV IE1 P13202Chromatin tethering motif M. leprae DNA helicase P40832 HhH bindingmotif

In yet other embodiments, the one or more heterologous domains can be anepigenetic modification domain. Non-limiting examples of suitableepigenetic modification domains include those with DNA deamination(e.g., cytidine deaminase, adenosine deaminase, guanine deaminase), DNAmethyltransferase activity (e.g., cytosine methyltransferase), DNAdemethylase activity, DNA amination, DNA oxidation activity, DNAhelicase activity, histone acetyltransferase (HAT) activity (e.g., HATdomain derived from E1A binding protein p300), histone deacetylaseactivity, histone methyltransferase activity, histone demethylaseactivity, histone kinase activity, histone phosphatase activity, histoneubiquitin ligase activity, histone deubiquitinating activity, histoneadenylation activity, histone deadenylation activity, histoneSUMOylating activity, histone deSUMOylating activity, histoneribosylation activity, histone deribosylation activity, histonemyristoylation activity, histone demyristoylation activity, histonecitrullination activity, histone alkylation activity, histonedealkylation activity, or histone oxidation activity. In specificembodiments, the epigenetic modification domain can comprise cytidinedeaminase activity, adenosine deaminase activity, histoneacetyltransferase activity, or DNA methyltransferase activity.

In other embodiments, the one or more heterologous domains can be atranscriptional regulation domain (i.e., a transcriptional activationdomain or transcriptional repressor domain). Suitable transcriptionalactivation domains include, without limit, herpes simplex virus VP16domain, VP64 (i.e., four tandem copies of VP16), VP160 (i.e., ten tandemcopies of VP16), NFκB p65 activation domain (p65), Epstein-Barr virus Rtransactivator (Rta) domain, VPR (i.e., VP64+p65+Rta), p300-dependenttranscriptional activation domains, p53 activation domains 1 and 2,heat-shock factor 1 (HSF1) activation domains, Smad4 activation domains(SAD), cAMP response element binding protein (CREB) activation domains,E2A activation domains, nuclear factor of activated T-cells (NFAT)activation domains, or combinations thereof. Non-limiting examples ofsuitable transcriptional repressor domains include Kruppel-associatedbox (KRAB) repressor domains, Mxi repressor domains, inducible cAMPearly repressor (ICER) domains, YY1 glycine rich repressor domains,Sp1-like repressors, E(spl) repressors, IκB repressors, Sin3 repressors,methyl-CpG binding protein 2 (MeCP2) repressors, or combinationsthereof. Transcriptional activation or transcriptional repressor domainscan be genetically fused to the Cas9 protein or bound via noncovalentprotein-protein, protein-RNA, or protein-DNA interactions.

In further embodiments, the one or more heterologous domains can be anRNA aptamer binding domain (Konermann et al., Nature, 2015,517(7536):583-588; Zalatan et al., Cell, 2015, 160(1-2):339-50).Examples of suitable RNA aptamer protein domains include MS2 coatprotein (MCP), PP7 bacteriophage coat protein (PCP), Mu bacteriophageCom protein, lambda bacteriophage N22 protein, stem-loop binding protein(SLBP), Fragile X mental retardation syndrome-related protein 1 (FXR1),proteins derived from bacteriophage such as AP205, BZ13, f1, f2, fd, fr,ID2, JP34/GA, JP501, JP34, JP500, KU1, M11, M12, MX1, NL95, PP7, ϕCb5,ϕCb8r, ϕCb12r, ϕCb23r, Qβ, R17, SP-β, TW18, TW19, and VK, fragmentsthereof, or derivatives thereof.

In yet other embodiments, the one or more heterologous domains can be anon-Cas9 nuclease domain. Suitable nuclease domains can be obtained fromany endonuclease or exonuclease. Non-limiting examples of endonucleasesfrom which a nuclease domain can be derived include, but are not limitedto, restriction endonucleases and homing endonucleases. In someembodiments, the nuclease domain can be derived from a type II-Srestriction endonuclease. Type II-S endonucleases cleave DNA at sitesthat are typically several base pairs away from the recognition/bindingsite and, as such, have separable binding and cleavage domains. Theseenzymes generally are monomers that transiently associate to form dimersto cleave each strand of DNA at staggered locations. Non-limitingexamples of suitable type II-S endonucleases include BfiI, BpmI, BsaI,BsgI, BsmBI, BsmI, BspMI, FokI, MboII, and SapI. In some embodiments,the nuclease domain can be a FokI nuclease domain or a derivativethereof. The type II-S nuclease domain can be modified to facilitatedimerization of two different nuclease domains. For example, thecleavage domain of FokI can be modified by mutating certain amino acidresidues. By way of non-limiting example, amino acid residues atpositions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499,500, 531, 534, 537, and 538 of FokI nuclease domains are targets formodification. In specific embodiments, the FokI nuclease domain cancomprise a first FokI half-domain comprising Q486E, I499L, and/or N496Dmutations, and a second FokI half-domain comprising E490K, I538K, and/orH537R mutations.

The one or more heterologous domains can be linked directly to the Cas9protein via one or more chemical bonds (e.g., covalent bonds), or theone or more heterologous domains can be linked indirectly to the Cas9protein via one or more linkers.

A linker is a chemical group that connects one or more other chemicalgroups via at least one covalent bond. Suitable linkers include aminoacids, peptides, nucleotides, nucleic acids, organic linker molecules(e.g., maleimide derivatives, N-ethoxybenzylimidazole,biphenyl-3,4′,5-tricarboxylic acid, p-aminobenzyloxycarbonyl, and thelike), disulfide linkers, and polymer linkers (e.g., PEG). The linkercan include one or more spacing groups including, but not limited toalkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl,heteroaryl, aralkyl, aralkenyl, aralkynyl and the like. The linker canbe neutral, or carry a positive or negative charge. Additionally, thelinker can be cleavable such that the linker's covalent bond thatconnects the linker to another chemical group can be broken or cleavedunder certain conditions, including pH, temperature, salt concentration,light, a catalyst, or an enzyme. In some embodiments, the linker can bea peptide linker. The peptide linker can be a flexible amino acid linker(e.g., comprising small, non-polar or polar amino acids). Non-limitingexamples of flexible linkers include LEGGGS (SEQ ID NO:108), TGSG (SEQID NO:109), GGSGGGSG (SEQ ID NO:110), (GGGGS)₁₋₄ (SEQ ID NO:111), and(Gly)₆₋₈ (SEQ ID NO:112). Alternatively, the peptide linker can be arigid amino acid linker. Such linkers include (EAAAK)₁₋₄ (SEQ IDNO:113), A(EAAAK)₂₋₅A (SEQ ID NO:114), PAPAP (SEQ ID NO:115), and(AP)₆₋₈ (SEQ ID NO:116). Additional examples of suitable linkers arewell known in the art and programs to design linkers are readilyavailable (Crasto et al., Protein Eng., 2000, 13(5):309-312).

In some embodiments, the engineered Cas9 proteins can be producedrecombinantly in cell-free systems, bacterial cells, or eukaryotic cellsand purified using standard purification means. In other embodiments,the engineered Cas9 proteins are produced in vivo in eukaryotic cells ofinterest from nucleic acids encoding the engineered Cas9 proteins (seesection (II) below).

In embodiments in which the engineered Cas9 protein comprises nucleaseor nickase activity, the engineered Cas9 protein can further comprise atleast one nuclear localization signal, cell-penetrating domain, and/ormarker domain, as well as at least one chromatin disrupting domain. Inembodiments in which the engineered Cas9 protein is linked to anepigenetic modification domain, the engineered Cas9 protein can furthercomprise at least one nuclear localization signal, cell-penetratingdomain, and/or marker domain, as well as at least one chromatindisrupting domain. Furthermore, in embodiments in which the engineeredCas9 protein is linked to a transcriptional regulation domain, theengineered Cas9 protein can further comprise at least one nuclearlocalization signal, cell-penetrating domain, and/or marker domain, aswell as at least one chromatin disrupting domain and/or at least one RNAaptamer binding domain.

(ii) Specific Engineered Cas9 Proteins

In specific embodiments, the engineered Cas9 protein is from Bacillussmithii, Lactobacillus rhamnosus, Parasutterella excrementihominis,Mycoplasma canis, Mycoplasma gallisepticum, Akkermansia glycaniphila,Akkermansia muciniphila, Oenococcus kitaharae, Bifidobacterium bombi,Acidothermus cellulolyticus, Alicyclobacillus hesperidum, Wolinellasuccinogenes, Nitratifractor salsuginis, Ralstonia syzygii, orCorynebacterium diphtheria and is linked to at least one NLS. In someiterations, the engineered Cas9 protein can have at least about 75%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, or at least about 99% sequence identity to SEQ ID NO:2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30. In certain embodiments,the engineered Cas9 protein can have at least about 95% sequenceidentity to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, or 30. In other iterations, the engineered Cas9 protein has theamino acid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, or 30.

In other embodiments, the engineered Cas9 protein can be a Bacillussmithii, Lactobacillus rhamnosus, Parasutterella excrementihominis,Mycoplasma canis, Mycoplasma gallisepticum, Akkermansia glycaniphila,Akkermansia muciniphila, Oenococcus kitaharae, Bifidobacterium bombi,Acidothermus cellulolyticus, Alicyclobacillus hesperidum, Wolinellasuccinogenes, Nitratifractor salsuginis, Ralstonia syzygii, orCorynebacterium diphtheria Cas9 protein linked to at least one chromatinmodulating motif (CMM). The linkage between the Cas9 protein and the CMMcan be direct or via a linker. The Cas9-CMM fusion protein can furthercomprise at least one NLS. In particular embodiments, the Cas9-CMMfusion protein can have at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or at least about 99%sequence identity to SEQ ID NO:117, 118, 119, 120, 121, 122, 123, or124. In certain embodiments, the Cas9-CMM fusion protein can have atleast about 95% sequence identity to SEQ ID NO:117, 118, 119, 120, 121,122, 123, or 124. In specific iterations, the Cas9-CMM fusion proteinhas the amino acid sequence of SEQ ID NO:117, 118, 119, 120, 121, 122,123, or 124.

(b) Engineered Guide RNAs

The engineered guide RNA is designed to complex with a specificengineered Cas9 protein. A guide RNA comprises (i) a CRISPR RNA (crRNA)that contains a guide sequence at the 5′ end that hybridizes with atarget sequence and (ii) a transacting crRNA (tracrRNA) sequence thatrecruits the Cas9 protein. The crRNA guide sequence of each guide RNA isdifferent (i.e., is sequence specific). The tracrRNA sequence isgenerally the same in guide RNAs designed to complex with a Cas9 proteinfrom a particular bacterial species.

The crRNA guide sequence is designed to hybridize with a target sequence(i.e., protospacer) in a double-stranded sequence. In general, thecomplementarity between the crRNA and the target sequence is at least80%, at least 85%, at least 90%, at least 95%, or at least 99%. Inspecific embodiments, the complementarity is complete (i.e., 100%). Invarious embodiments, the length of the crRNA guide sequence can rangefrom about 15 nucleotides to about 25 nucleotides. For example, thecrRNA guide sequence can be about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, or 25 nucleotides in length. In specific embodiments, the crRNA isabout 19, 20, or 21 nucleotides in length. In one embodiment, the crRNAguide sequence has a length of 20 nucleotides.

The guide RNA comprises repeat sequence that forms at least one stemloop structure, which interacts with the Cas9 protein, and 3′ sequencethat remains single-stranded. The length of each loop and stem can vary.For example, the loop can range from about 3 to about 10 nucleotides inlength, and the stem can range from about 6 to about 20 base pairs inlength. The stem can comprise one or more bulges of 1 to about 10nucleotides. The length of the single-stranded 3′ region can vary. ThetracrRNA sequence in the engineered guide RNA generally is based uponthe coding sequence of wild type tracrRNA in the bacterial species ofinterest. The wild-type sequence can be modified to facilitate secondarystructure formation, increased secondary structure stability, facilitateexpression in eukaryotic cells, and so forth. For example, one or morenucleotide changes can be introduced into the guide RNA coding sequence(see Example 3, below). The tracrRNA sequence can range in length fromabout 50 nucleotides to about 300 nucleotides. In various embodiments,the tracrRNA can range in length from about 50 to about 90 nucleotides,from about 90 to about 110 nucleotides, from about 110 to about 130nucleotides, from about 130 to about 150 nucleotides, from about 150 toabout 170 nucleotides, from about 170 to about 200 nucleotides, fromabout 200 to about 250 nucleotides, or from about 250 to about 300nucleotides.

In general, the engineered guide RNA is a single molecule (i.e., asingle guide RNA or sgRNA), wherein the crRNA sequence is linked to thetracrRNA sequence. In some embodiments, however, the engineered guideRNA can be two separate molecules. A first molecule comprising the crRNAthat contains 3′ sequence (comprising from about 6 to about 20nucleotides) that is capable of base pairing with the 5′ end of a secondmolecule, wherein the second molecule comprises the tracrRNA thatcontains 5′ sequence (comprising from about 6 to about 20 nucleotides)that is capable of base pairing with the 3′ end of the first molecule.

In some embodiments, the tracrRNA sequence of the engineered guide RNAcan be modified to comprise one or more aptamer sequences (Konermann etal., Nature, 2015, 517(7536):583-588; Zalatan et al., Cell, 2015,160(1-2):339-50). Suitable aptamer sequences include those that bindadaptor proteins chosen from MCP, PCP, Com, SLBP, FXR1, AP205, BZ13, f1,f2, fd, fr, ID2, JP34/GA, JP501, JP34, JP500, KU1, M11, M12, MX1, NL95,PP7, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, Qβ, R17, SP-β, TW18, TW19, VK,fragments thereof, or derivatives thereof. Those of skill in the artappreciate that the length of the aptamer sequence can vary.

In other embodiments, the guide RNA can further comprise at least onedetectable label. The detectable label can be a fluorophore (e.g., FAM,TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags, orsuitable fluorescent dye), a detection tag (e.g., biotin, digoxigenin,and the like), quantum dots, or gold particles.

The guide RNA can comprise standard ribonucleotides and/or modifiedribonucleotides. In some embodiment, the guide RNA can comprise standardor modified deoxyribonucleotides. In embodiments in which the guide RNAis enzymatically synthesized (i.e., in vivo or in vitro), the guide RNAgenerally comprises standard ribonucleotides. In embodiments in whichthe guide RNA is chemically synthesized, the guide RNA can comprisestandard or modified ribonucleotides and/or deoxyribonucleotides.Modified ribonucleotides and/or deoxyribonucleotides include basemodifications (e.g., pseudouridine, 2-thiouridine, N6-methyladenosine,and the like) and/or sugar modifications (e.g., 2′-O-methy, 2′-fluoro,2′-amino, locked nucleic acid (LNA), and so forth). The backbone of theguide RNA can also be modified to comprise phosphorothioate linkages,boranophosphate linkages, or peptide nucleic acids.

In specific embodiments, the engineered guide RNA has at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or at least about 99% sequence identity to SEQ IDNO:31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45. Insome embodiments, the engineered Cas9 guide RNA has the sequence of SEQID NO:31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45.

(c) PAM Sequence

The engineered Cas9 systems detailed above target specific sequences indouble-stranded DNA that are located upstream of novel PAM sequences.The PAM sequences preferred by the engineered Cas9 systems wereidentified in vitro using a library of degenerate PAMS (see Example 1and FIG. 1), and confirmed by sequencing after genome editingexperiments (see Example 2). The PAM for each of the engineered Cas9system disclosed herein is presented in Table A, below.

TABLE A PAM Sequences PAM Engineered Cas9system (5′-3′)*Bacillus smithii Cas9 (BsmCas9) NNNNCAAALactobacillus rhamnosus Cas9 (LrhCas9) NGAAAParasutterella excrementihominis Cas9 NGG (PexCas9)Mycoplasma canis Cas9 (McaCas9) NNGGMycoplasma gaffisepticum Cas9 (MgaCas9) NNAATAkkermansia glycaniphila Cas9 (AgICas9) NNNRTAAkkermansia muciniphila Cas9 (AmuCas9) MMACCAOenococcus kitaharae Cas9 (OkiCas9) NNGBifidobacterium bombi Cas9 (BboCas9) NNNNGRYAcidothermus cellulolyticus Cas9 NGG (AceCas9)Alicyclobacillus hesperidum Cas9 NGG (AheCas9)Wolinella succinogenes Cas9 (WsuCas9) NGG Nitratifractor salsuginis Cas9NRGNK (NsaCas9) Ralstonia syzygfi Cas9 (RsyCas9) GGGRGCorynebacterium diphtheria Cas9 NNAMMMC (CdiCas9) *K is G or T; M is Aor C; R is A or G; Y is C or T; and N is A, C, G, or T.(II) Nucleic Acids

A further aspect of the present disclosure provides nucleic acidsencoding the engineered Cas9 systems described above in section (I). Thesystems can be encoded by single nucleic acids or multiple nucleicacids. The nucleic acids can be DNA or RNA, linear or circular,single-stranded or double-stranded. The RNA or DNA can be codonoptimized for efficient translation into protein in the eukaryotic cellof interest. Codon optimization programs are available as freeware orfrom commercial sources.

In some embodiments, nucleic acid encodes a protein having at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, or at least about 99% sequence identity to the aminoacid sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, or 30. In certain embodiments, the nucleic acid encoding theengineered Cas9 protein can have at least about 75%, at least about 80%,at least about 85%, at least about 90%, at least about 95%, or at leastabout 99% sequence identity to the DNA sequence of SEQ ID NO:1, 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29. In certain embodiments,the DNA encoding the engineered Cas9 protein has the DNA sequence of SEQID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, or 29. Inadditional embodiments, the nucleic acid encodes a protein having atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 99% sequence identity to theamino acid sequence of SEQ ID NO:117, 118, 119, 120, 121, 122, 123, or124.

In some embodiments, the nucleic acid encoding the engineered Cas9protein can be RNA. The RNA can be enzymatically synthesized in vitro.For this, DNA encoding the engineered Cas9 protein can be operablylinked to a promoter sequence that is recognized by a phage RNApolymerase for in vitro RNA synthesis. For example, the promotersequence can be a T7, T3, or SP6 promoter sequence or a variation of aT7, T3, or SP6 promoter sequence. The DNA encoding the engineeredprotein can be part of a vector, as detailed below. In such embodiments,the in vitro-transcribed RNA can be purified, capped, and/orpolyadenylated. In other embodiments, the RNA encoding the engineeredCas9 protein can be part of a self-replicating RNA (Yoshioka et al.,Cell Stem Cell, 2013, 13:246-254). The self-replicating RNA can bederived from a noninfectious, self-replicating Venezuelan equineencephalitis (VEE) virus RNA replicon, which is a positive-sense,single-stranded RNA that is capable of self-replicating for a limitednumber of cell divisions, and which can be modified to code proteins ofinterest (Yoshioka et al., Cell Stem Cell, 2013, 13:246-254).

In other embodiments, the nucleic acid encoding the engineered Cas9protein can be DNA. The DNA coding sequence can be operably linked to atleast one promoter control sequence for expression in the cell ofinterest. In certain embodiments, the DNA coding sequence can beoperably linked to a promoter sequence for expression of the engineeredCas9 protein in bacterial (e.g., E. coli) cells or eukaryotic (e.g.,yeast, insect, or mammalian) cells. Suitable bacterial promotersinclude, without limit, T7 promoters, lac operon promoters, trppromoters, tac promoters (which are hybrids of trp and lac promoters),variations of any of the foregoing, and combinations of any of theforegoing. Non-limiting examples of suitable eukaryotic promotersinclude constitutive, regulated, or cell- or tissue-specific promoters.Suitable eukaryotic constitutive promoter control sequences include, butare not limited to, cytomegalovirus immediate early promoter (CMV),simian virus (SV40) promoter, adenovirus major late promoter, Roussarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter,phosphoglycerate kinase (PGK) promoter, elongation factor (ED1)-alphapromoter, ubiquitin promoters, actin promoters, tubulin promoters,immunoglobulin promoters, fragments thereof, or combinations of any ofthe foregoing. Examples of suitable eukaryotic regulated promotercontrol sequences include without limit those regulated by heat shock,metals, steroids, antibiotics, or alcohol. Non-limiting examples oftissue-specific promoters include B29 promoter, CD14 promoter, CD43promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAPpromoter, GPIlb promoter, ICAM-2 promoter, INF-β promoter, Mb promoter,NphsI promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASPpromoter. The promoter sequence can be wild type or it can be modifiedfor more efficient or efficacious expression. In some embodiments, theDNA coding sequence also can be linked to a polyadenylation signal(e.g., SV40 polyA signal, bovine growth hormone (BGH) polyA signal,etc.) and/or at least one transcriptional termination sequence. In somesituations, the engineered Cas9 protein can be purified from thebacterial or eukaryotic cells.

In still other embodiments, the engineered guide RNA can be encoded byDNA. In some instances, the DNA encoding the engineered guide RNA can beoperably linked to a promoter sequence that is recognized by a phage RNApolymerase for in vitro RNA synthesis. For example, the promotersequence can be a T7, T3, or SP6 promoter sequence or a variation of aT7, T3, or SP6 promoter sequence. In other instances, the DNA encodingthe engineered guide RNA can be operably linked to a promoter sequencethat is recognized by RNA polymerase III (Pol III) for expression ineukaryotic cells of interest. Examples of suitable Pol III promotersinclude, but are not limited to, mammalian U6, U3, H1, and 7SL RNApromoters.

In various embodiments, the nucleic acid encoding the engineered Cas9protein can be present in a vector. In some embodiments, the vector canfurther comprise nucleic acid encoding the engineered guide RNA.Suitable vectors include plasmid vectors, viral vectors, andself-replicating RNA (Yoshioka et al., Cell Stem Cell, 2013,13:246-254). In some embodiments, the nucleic acid encoding the complexor fusion protein can be present in a plasmid vector. Non-limitingexamples of suitable plasmid vectors include pUC, pBR322, pET,pBluescript, and variants thereof. In other embodiments, the nucleicacid encoding the complex or fusion protein can be part of a viralvector (e.g., lentiviral vectors, adeno-associated viral vectors,adenoviral vectors, and so forth). The plasmid or viral vector cancomprise additional expression control sequences (e.g., enhancersequences, Kozak sequences, polyadenylation sequences, transcriptionaltermination sequences, etc.), selectable marker sequences (e.g.,antibiotic resistance genes), origins of replication, and the like.Additional information about vectors and use thereof can be found in“Current Protocols in Molecular Biology” Ausubel et al., John Wiley &Sons, New York, 2003 or “Molecular Cloning: A Laboratory Manual”Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,3rd edition, 2001.

(III) Eukaryotic Cells

Another aspect of the present disclosure comprises eukaryotic cellscomprising at least one engineered Cas9 system as detailed above insection (I) and/or at least one nucleic acid encoding an engineered Cas9protein and/or engineered guide RNA as detailed above in section (II).

The eukaryotic cell can be a human cell, a non-human mammalian cell, anon-mammalian vertebrate cell, an invertebrate cell, a plant cell, or asingle cell eukaryotic organism. Examples of suitable eukaryotic cellsare detailed below in section (IV)(c). The eukaryotic cell can be invitro, ex vivo, or in vivo.

(IV) Methods for Modifying Chromosomal Sequences

A further aspect of the present disclosure encompasses methods formodifying a chromosomal sequence in eukaryotic cells. In general, themethods comprise introducing into the eukaryotic cell of interest atleast one engineered Cas9 system as detailed above in section (I) and/orat least one nucleic acid encoding said engineered Cas9 system asdetailed above in section (II).

In embodiments in which the engineered Cas9 protein comprises nucleaseor nickase activity, the chromosomal sequence modification can comprisea substitution of at least one nucleotide, a deletion of at least onenucleotide, an insertion of at least one nucleotide. In some iterations,the method comprises introducing into the eukaryotic cell one engineeredCas9 system comprising nuclease activity or two engineered Cas9 systemscomprising nickase activity and no donor polynucleotide, such that theengineered Cas9 system or systems introduce a double-stranded break inthe target site in the chromosomal sequence and repair of thedouble-stranded break by cellular DNA repair processes introduces atleast one nucleotide change (i.e., indel), thereby inactivating thechromosomal sequence (i.e., gene knock-out). In other iterations, themethod comprises introducing into the eukaryotic cell one engineeredCas9 system comprising nuclease activity or two engineered Cas9 systemscomprising nickase activity, as well as the donor polynucleotide, suchthat the engineered Cas9 system or systems introduce a double-strandedbreak in the target site in the chromosomal sequence and repair of thedouble-stranded break by cellular DNA repair processes leads toinsertion or exchange of sequence in the donor polynucleotide into thetarget site in the chromosomal sequence (i.e., gene correction or geneknock-in).

In embodiments, in which the engineered Cas9 protein comprisesepigenetic modification activity or transcriptional regulation activity,the chromosomal sequence modification can comprise a conversion of atleast one nucleotide in or near the target site, a modification of atleast one nucleotide in or near the target site, a modification of atleast one histone protein in or near the target site, and/or a change intranscription in or near the target site in the chromosomal sequence.

(a) Introduction into the Cell

As mentioned above, the method comprises introducing into the eukaryoticcell at least one engineered Cas9 system and/or nucleic acid encodingsaid system (and optional donor polynucleotide). The at least one systemand/or nucleic acid/donor polynucleotide can be introduced into the cellof interest by a variety of means.

In some embodiments, the cell can be transfected with the appropriatemolecules (i.e., protein, DNA, and/or RNA). Suitable transfectionmethods include nucleofection (or electroporation), calciumphosphate-mediated transfection, cationic polymer transfection (e.g.,DEAE-dextran or polyethylenimine), viral transduction, virosometransfection, virion transfection, liposome transfection, cationicliposome transfection, immunoliposome transfection, nonliposomal lipidtransfection, dendrimer transfection, heat shock transfection,magnetofection, lipofection, gene gun delivery, impalefection,sonoporation, optical transfection, and proprietary agent-enhanceduptake of nucleic acids. Transfection methods are well known in the art(see, e.g., “Current Protocols in Molecular Biology” Ausubel et al.,John Wiley & Sons, New York, 2003 or “Molecular Cloning: A LaboratoryManual” Sambrook & Russell, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., 3rd edition, 2001). In other embodiments, the moleculescan be introduced into the cell by microinjection. For example, themolecules can be injected into the cytoplasm or nuclei of the cells ofinterest. The amount of each molecule introduced into the cell can vary,but those skilled in the art are familiar with means for determining theappropriate amount.

The various molecules can be introduced into the cell simultaneously orsequentially. For example, the engineered Cas9 system (or its encodingnucleic acid) and the donor polynucleotide can be introduced at the sametime. Alternatively, one can be introduced first and then the other canbe introduced later into the cell.

In general, the cell is maintained under conditions appropriate for cellgrowth and/or maintenance. Suitable cell culture conditions are wellknown in the art and are described, for example, in Santiago et al.,Proc. Natl. Acad. Sci. USA, 2008, 105:5809-5814; Moehle et al. Proc.Natl. Acad. Sci. USA, 2007, 104:3055-3060; Urnov et al., Nature, 2005,435:646-651; and Lombardo et al., Nat. Biotechnol., 2007, 25:1298-1306.Those of skill in the art appreciate that methods for culturing cellsare known in the art and can and will vary depending on the cell type.Routine optimization may be used, in all cases, to determine the besttechniques for a particular cell type.

(b) Optional Donor Polynucleotide

In embodiments in which the engineered Cas9 protein comprises nucleaseor nickase activity, the method can further comprise introducing atleast one donor polynucleotide into the cell. The donor polynucleotidecan be single-stranded or double-stranded, linear or circular, and/orRNA or DNA. In some embodiments, the donor polynucleotide can be avector, e.g., a plasmid vector.

The donor polynucleotide comprises at least one donor sequence. In someaspects, the donor sequence of the donor polynucleotide can be amodified version of an endogenous or native chromosomal sequence. Forexample, the donor sequence can be essentially identical to a portion ofthe chromosomal sequence at or near the sequence targeted by theengineered Cas9 system, but which comprises at least one nucleotidechange. Thus, upon integration or exchange with the native sequence, thesequence at the targeted chromosomal location comprises at least onenucleotide change. For example, the change can be an insertion of one ormore nucleotides, a deletion of one or more nucleotides, a substitutionof one or more nucleotides, or combinations thereof. As a consequence ofthe “gene correction” integration of the modified sequence, the cell canproduce a modified gene product from the targeted chromosomal sequence.

In other aspects, the donor sequence of the donor polynucleotide can bean exogenous sequence. As used herein, an “exogenous” sequence refers toa sequence that is not native to the cell, or a sequence whose nativelocation is in a different location in the genome of the cell. Forexample, the exogenous sequence can comprise protein coding sequence,which can be operably linked to an exogenous promoter control sequencesuch that, upon integration into the genome, the cell is able to expressthe protein coded by the integrated sequence. Alternatively, theexogenous sequence can be integrated into the chromosomal sequence suchthat its expression is regulated by an endogenous promoter controlsequence. In other iterations, the exogenous sequence can be atranscriptional control sequence, another expression control sequence,an RNA coding sequence, and so forth. As noted above, integration of anexogenous sequence into a chromosomal sequence is termed a “knock in.”

As can be appreciated by those skilled in the art, the length of thedonor sequence can and will vary. For example, the donor sequence canvary in length from several nucleotides to hundreds of nucleotides tohundreds of thousands of nucleotides.

Typically, the donor sequence in the donor polynucleotide is flanked byan upstream sequence and a downstream sequence, which have substantialsequence identity to sequences located upstream and downstream,respectively, of the sequence targeted by the engineered Cas9 system.Because of these sequence similarities, the upstream and downstreamsequences of the donor polynucleotide permit homologous recombinationbetween the donor polynucleotide and the targeted chromosomal sequencesuch that the donor sequence can be integrated into (or exchanged with)the chromosomal sequence.

The upstream sequence, as used herein, refers to a nucleic acid sequencethat shares substantial sequence identity with a chromosomal sequenceupstream of the sequence targeted by the engineered Cas9 system.Similarly, the downstream sequence refers to a nucleic acid sequencethat shares substantial sequence identity with a chromosomal sequencedownstream of the sequence targeted by the engineered Cas9 system. Asused herein, the phrase “substantial sequence identity” refers tosequences having at least about 75% sequence identity. Thus, theupstream and downstream sequences in the donor polynucleotide can haveabout 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% A sequenceidentity with sequence upstream or downstream to the target sequence. Inan exemplary embodiment, the upstream and downstream sequences in thedonor polynucleotide can have about 95% or 100% sequence identity withchromosomal sequences upstream or downstream to the sequence targeted bythe engineered Cas9 system.

In some embodiments, the upstream sequence shares substantial sequenceidentity with a chromosomal sequence located immediately upstream of thesequence targeted by the engineered Cas9 system. In other embodiments,the upstream sequence shares substantial sequence identity with achromosomal sequence that is located within about one hundred (100)nucleotides upstream from the target sequence. Thus, for example, theupstream sequence can share substantial sequence identity with achromosomal sequence that is located about 1 to about 20, about 21 toabout 40, about 41 to about 60, about 61 to about 80, or about 81 toabout 100 nucleotides upstream from the target sequence. In someembodiments, the downstream sequence shares substantial sequenceidentity with a chromosomal sequence located immediately downstream ofthe sequence targeted by the engineered Cas9 system. In otherembodiments, the downstream sequence shares substantial sequenceidentity with a chromosomal sequence that is located within about onehundred (100) nucleotides downstream from the target sequence. Thus, forexample, the downstream sequence can share substantial sequence identitywith a chromosomal sequence that is located about 1 to about 20, about21 to about 40, about 41 to about 60, about 61 to about 80, or about 81to about 100 nucleotides downstream from the target sequence.

Each upstream or downstream sequence can range in length from about 20nucleotides to about 5000 nucleotides. In some embodiments, upstream anddownstream sequences can comprise about 50, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2800, 3000, 3200,3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, or 5000 nucleotides. Inspecific embodiments, upstream and downstream sequences can range inlength from about 50 to about 1500 nucleotides.

(c) Cell Types

A variety of eukaryotic cells are suitable for use in the methodsdisclosed herein. For example, the cell can be a human cell, a non-humanmammalian cell, a non-mammalian vertebrate cell, an invertebrate cell,an insect cell, a plant cell, a yeast cell, or a single cell eukaryoticorganism. In some embodiments, the cell can be a one cell embryo. Forexample, a non-human mammalian embryo including rat, hamster, rodent,rabbit, feline, canine, ovine, porcine, bovine, equine, and primateembryos. In still other embodiments, the cell can be a stem cell such asembryonic stem cells, ES-like stem cells, fetal stem cells, adult stemcells, and the like. In one embodiment, the stem cell is not a humanembryonic stem cell. Furthermore, the stem cells may include those madeby the techniques disclosed in WO2003/046141, which is incorporatedherein in its entirety, or Chung et al. (Cell Stem Cell, 2008,2:113-117). The cell can be in vitro (i.e., in culture), ex vivo (i.e.,within tissue isolated from an organism), or in vivo (i.e., within anorganism). In exemplary embodiments, the cell is a mammalian cell ormammalian cell line. In particular embodiments, the cell is a human cellor human cell line.

Non-limiting examples of suitable mammalian cells or cell lines includehuman embryonic kidney cells (HEK293, HEK293T); human cervical carcinomacells (HELA); human lung cells (W138); human liver cells (Hep G2); humanU2-OS osteosarcoma cells, human A549 cells, human A-431 cells, and humanK562 cells; Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK)cells; mouse myeloma NSO cells, mouse embryonic fibroblast 3T3 cells(NIH3T3), mouse B lymphoma A20 cells; mouse melanoma B16 cells; mousemyoblast C2C12 cells; mouse myeloma SP2/0 cells; mouse embryonicmesenchymal C3H-10T1/2 cells; mouse carcinoma CT26 cells, mouse prostateDuCuP cells; mouse breast EMT6 cells; mouse hepatoma Nepa1c1c7 cells;mouse myeloma J5582 cells; mouse epithelial MTD-1A cells; mousemyocardial MyEnd cells; mouse renal RenCa cells; mouse pancreatic RIN-5Fcells; mouse melanoma X64 cells; mouse lymphoma YAC-1 cells; ratglioblastoma 9L cells; rat B lymphoma RBL cells; rat neuroblastoma B35cells; rat hepatoma cells (HTC); buffalo rat liver BRL 3A cells; caninekidney cells (MDCK); canine mammary (CMT) cells; rat osteosarcoma D17cells; rat monocyte/macrophage DH82 cells; monkey kidney SV-40transformed fibroblast (COS7) cells; monkey kidney CVI-76 cells; Africangreen monkey kidney (VERO-76) cells. An extensive list of mammalian celllines may be found in the American Type Culture Collection catalog(ATCC, Manassas, Va.).

(V) Applications

The compositions and methods disclosed herein can be used in a varietyof therapeutic, diagnostic, industrial, and research applications. Insome embodiments, the present disclosure can be used to modify anychromosomal sequence of interest in a cell, animal, or plant in order tomodel and/or study the function of genes, study genetic or epigeneticconditions of interest, or study biochemical pathways involved invarious diseases or disorders. For example, transgenic organisms can becreated that model diseases or disorders, wherein the expression of oneor more nucleic acid sequences associated with a disease or disorder isaltered. The disease model can be used to study the effects of mutationson the organism, study the development and/or progression of thedisease, study the effect of a pharmaceutically active compound on thedisease, and/or assess the efficacy of a potential gene therapystrategy.

In other embodiments, the compositions and methods can be used toperform efficient and cost effective functional genomic screens, whichcan be used to study the function of genes involved in a particularbiological process and how any alteration in gene expression can affectthe biological process, or to perform saturating or deep scanningmutagenesis of genomic loci in conjunction with a cellular phenotype.Saturating or deep scanning mutagenesis can be used to determinecritical minimal features and discrete vulnerabilities of functionalelements required for gene expression, drug resistance, and reversal ofdisease, for example.

In further embodiments, the compositions and methods disclosed hereincan be used for diagnostic tests to establish the presence of a diseaseor disorder and/or for use in determining treatment options. Examples ofsuitable diagnostic tests include detection of specific mutations incancer cells (e.g., specific mutation in EGFR, HER2, and the like),detection of specific mutations associated with particular diseases(e.g., trinucleotide repeats, mutations in β-globin associated withsickle cell disease, specific SNPs, etc.), detection of hepatitis,detection of viruses (e.g., Zika), and so forth.

In additional embodiments, the compositions and methods disclosed hereincan be used to correct genetic mutations associated with a particulardisease or disorder such as, e.g., correct globin gene mutationsassociated with sickle cell disease or thalassemia, correct mutations inthe adenosine deaminase gene associated with severe combined immunedeficiency (SCID), reduce the expression of HTT, the disease-causinggene of Huntington's disease, or correct mutations in the rhodopsin genefor the treatment of retinitis pigmentosa. Such modifications may bemade in cells ex vivo.

In still other embodiments, the compositions and methods disclosedherein can be used to generate crop plants with improved traits orincreased resistance to environmental stresses. The present disclosurecan also be used to generate farm animal with improved traits orproduction animals. For example, pigs have many features that make themattractive as biomedical models, especially in regenerative medicine orxenotransplantation.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd Ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

When introducing elements of the present disclosure or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

The term “about” when used in relation to a numerical value, x, forexample means x±5%.

As used herein, the terms “complementary” or “complementarity” refer tothe association of double-stranded nucleic acids by base pairing throughspecific hydrogen bonds. The base pairing may be standard Watson-Crickbase pairing (e.g., 5′-A G T C-3′ pairs with the complementary sequence3′-T C A G-5′). The base pairing also may be Hoogsteen or reversedHoogsteen hydrogen bonding. Complementarity is typically measured withrespect to a duplex region and thus, excludes overhangs, for example.Complementarity between two strands of the duplex region may be partialand expressed as a percentage (e.g., 70%), if only some (e.g., 70%) ofthe bases are complementary. The bases that are not complementary are“mismatched.” Complementarity may also be complete (i.e., 100%), if allthe bases in the duplex region are complementary.

As used herein, the term “CRISPR/Cas system” or “Cas9 system” refers toa complex comprising a Cas9 protein (i.e., nuclease, nickase, orcatalytically dead protein) and a guide RNA.

The term “endogenous sequence,” as used herein, refers to a chromosomalsequence that is native to the cell.

As used herein, the term “exogenous” refers to a sequence that is notnative to the cell, or a chromosomal sequence whose native location inthe genome of the cell is in a different chromosomal location.

A “gene,” as used herein, refers to a DNA region (including exons andintrons) encoding a gene product, as well as all DNA regions whichregulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites, and locus control regions.

The term “heterologous” refers to an entity that is not endogenous ornative to the cell of interest. For example, a heterologous proteinrefers to a protein that is derived from or was originally derived froman exogenous source, such as an exogenously introduced nucleic acidsequence. In some instances, the heterologous protein is not normallyproduced by the cell of interest.

The term “nickase” refers to an enzyme that cleaves one strand of adouble-stranded nucleic acid sequence (i.e., nicks a double-strandedsequence). For example, a nuclease with double strand cleavage activitycan be modified by mutation and/or deletion to function as a nickase andcleave only one strand of a double-stranded sequence.

The term “nuclease,” as used herein, refers to an enzyme that cleavesboth strands of a double-stranded nucleic acid sequence.

The terms “nucleic acid” and “polynucleotide” refer to adeoxyribonucleotide or ribonucleotide polymer, in linear or circularconformation, and in either single- or double-stranded form. For thepurposes of the present disclosure, these terms are not to be construedas limiting with respect to the length of a polymer. The terms canencompass known analogs of natural nucleotides, as well as nucleotidesthat are modified in the base, sugar and/or phosphate moieties (e.g.,phosphorothioate backbones). In general, an analog of a particularnucleotide has the same base-pairing specificity; i.e., an analog of Awill base-pair with T.

The term “nucleotide” refers to deoxyribonucleotides or ribonucleotides.The nucleotides may be standard nucleotides (i.e., adenosine, guanosine,cytidine, thymidine, and uridine), nucleotide isomers, or nucleotideanalogs. A nucleotide analog refers to a nucleotide having a modifiedpurine or pyrimidine base or a modified ribose moiety. A nucleotideanalog may be a naturally occurring nucleotide (e.g., inosine,pseudouridine, etc.) or a non-naturally occurring nucleotide.Non-limiting examples of modifications on the sugar or base moieties ofa nucleotide include the addition (or removal) of acetyl groups, aminogroups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methylgroups, phosphoryl groups, and thiol groups, as well as the substitutionof the carbon and nitrogen atoms of the bases with other atoms (e.g.,7-deaza purines). Nucleotide analogs also include dideoxy nucleotides,2′-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleicacids (PNA), and morpholinos.

The terms “polypeptide” and “protein” are used interchangeably to referto a polymer of amino acid residues.

The terms “target sequence,” “target chromosomal sequence,” and “targetsite” are used interchangeably to refer to the specific sequence inchromosomal DNA to which the engineered Cas9 system is targeted, and thesite at which the engineered Cas9 system modifies the DNA or protein(s)associated with the DNA.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. An approximatealignment for nucleic acid sequences is provided by the local homologyalgorithm of Smith and Waterman, Advances in Applied Mathematics2:482-489 (1981). This algorithm can be applied to amino acid sequencesby using the scoring matrix developed by Dayhoff, Atlas of ProteinSequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, NationalBiomedical Research Foundation, Washington, D.C., USA, and normalized byGribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplaryimplementation of this algorithm to determine percent identity of asequence is provided by the Genetics Computer Group (Madison, Wis.) inthe “BestFit” utility application. Other suitable programs forcalculating the percent identity or similarity between sequences aregenerally known in the art, for example, another alignment program isBLAST, used with default parameters. For example, BLASTN and BLASTP canbe used using the following default parameters: genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GENBANK+EMBL+DDBJ+PDB+GENBANK CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found on theGENBANK NIH genetic sequence database website.

As various changes could be made in the above-described cells andmethods without departing from the scope of the invention, it isintended that all matter contained in the above description and in theexamples given below, shall be interpreted as illustrative and not in alimiting sense.

EXAMPLES

The following examples illustrate certain aspects of the disclosure.

Example 1: Determination of PAM Requirements for Target DNA Cleavage byCas9 Orthologs

Cas9 orthologs from Bacillus smithfi, Lactobacillus rhamnosus,Parasutterella excrementihominis, Mycoplasma canis, Mycoplasmagallisepticum, Akkermansia glycaniphila, Akkermansia muciniphila,Oenococcus kitaharae, Bifidobacterium bombi, Acidothermuscellulolyticus, Alicyclobacillus hesperidum, Wolinella succinogenes,Nitratifractor salsuginis, Ralstonia syzygfi, and Corynebacteriumdiphtheria were codon optimized for expression in human cells and taggedwith a SV40 large T antigen nuclear localization (NLS) on the C terminus(SEQ ID NOs:1-30; see Table 6 below). The expression of each orthologwas driven by a human cytomegalovirus (CMV) immediate early enhancer andpromoter. CRISPR RNA (crRNA) and putative trans-activating crRNA(tracrRNA) for each ortholog were joined together to form a single guideRNA (sgRNA) (SEQ ID NOs:31-45; see Table 6 below). The expression ofeach sgRNA was driven by a human U6 promoter. In vitro transcribed sgRNAwas prepared from a T7 promoter tagged PCR template as a supplement forin vitro digestion.

Human K562 cells were transfected with Cas9 encoding plasmid and sgRNAexpression plasmid by nucleofection. Each transfection consisted of 2million cells, 5 μg of Cas9 encoding plasmid DNA, and 3 μg of sgRNAexpression plasmid DNA. Cells were harvested approximately 24 hr posttransfection, washed with ice cold PBS buffer, and lysed with 150 μL oflysis solution (20 mM HEPES, pH 7.5; 100 mM KCl; 5 mM MgCl2, 1 mM DTT,5% glycerol, 0.1% Triton X-100, 1×Protease inhibitor) with constantagitation for 30 minutes in a 4° C. cold room. Supernatant was preparedby removing residual cellular debris with centrifugation at 16,000×g for2 minutes at 4° C. and used as a source of Cas9 RNP for in vitrodigestion of a plasmid DNA PAM library. The library contained 4⁸degenerate PAMs, each immediately preceded by a protospacer with thefollowing configuration: 5′-GTACAAACGGCAGAAGCTGGNNNNNNNN-3′ (SEQ IDNO:46). Each in vitro digestion consisted of 10 μL of cell lysatesupernatant, 2 μL of 5× digestion buffer (100 mM HEPES, pH 7.5; 500 mMKCl; 25 mM MgCl2; 5 mM DTT; 25% glycerol), 800 ng of PAM library DNA,and 20 pmol of in vitro transcribed sgRNA supplement in a 20 μL reactionvolume. Reaction was maintained at 37° C. for 30 minutes and thenpurified with PCR purification kit. Illumina NextSeq sequencinglibraries were prepared from digested products and subjected to deepsequencing. Deep sequencing data were analyzed using a Weblogo programto deduce the PAM requirement for each Cas9 ortholog.

Results are summarized in FIG. 1. The results revealed several Cas9orthologs that use A and/or T containing PAMs for in vitro target DNAcleavage. These Cas9 orthologs could provide a means to target AT richgenomic sites. The results also revealed several Cas9 orthologs that usea PAM suitable for targeting GC rich genomic sites. These Cas9 orthologscould provide alternative targeting schemes to SpyCas9 in GC richgenomic sites to increase targeting resolution and specificity.

Example 2: Genome Modification Using Bacillus smithii Cas9 (BsmCas9) andLactobacillus rhamnosus Cas9 (LrhCas9)

As shown in FIG. 1 and Table A (above), the small BsmCas9 (1095 aa) (SEQID NO: 2) and the LrhCas9 (SEQ ID NO: 4) use a 5′-NNNNCAAA-3′ PAM and a5′-NGAAA-3′ PAM for target DNA binding, respectively. These novel PAMusages provide a means to target AT rich genomic sites. To demonstrategene editing, human K562 cells (1×10⁶) were nucleofected with 5 μg ofCas9 encoding plasmid DNA and 3 μg of sgRNA expression plasmid DNA.Targeted genomic sites include the human tyrosine-protein phosphatasenon-receptor type 2 (PTN2) locus, the human empty spiracles homeobox 1(EMX1) locus, the human programmed cell death 1 ligand 1 (PD1L1) locus,the human AAVS1 safe harbor locus, the human cytochrome p450oxidoreductase (POR) locus, and the human nuclear receptor subfamily 1group I member 3(CAR) locus. Genomic DNA was prepared using a DNAextraction solution (QuickExtract™) three days post transfection andtargeted genomic regions were each PCR amplified (JumpStart Tag™ReadyMix™). The PCR primers are listed in Table 1.

TABLE 1 PCR Primers. Forward Reverse Size Locus primer (5′-3′)primer (5′-3′) (bp) PTN2 CTGTTTCCTGGGTTCCA ACAAGGGCTCAAGTGGAGT 290ATAACAAGAC G (SEQ ID NO: 47) (SEQ ID NO: 48) EMX1 ATGGGAGCAGCTGGTCACAGCCCATTGCTTGTCCCT 507 GAG (SEQ ID NO: 50) (SEQ ID NO: 49) PD1L1CTCGCCATTCCAGCCAC GGTTAAGTCGGGTTTCCTT 341 TCAAAC GCAG (SEQ ID NO: 51)(SEQ ID NO: 52) AAVS1 TTCGGGTCACCTCTCAC GGCTCCATCGTAAGCAAAC 469 TCC C(SEQ ID NO: 53) (SEQ ID NO: 54) POR CTCCCCTGCTTCTTGTCACAGGTCGTGGACACTCAC 380 GTAT A (SEQ ID NO: 55) (SEQ ID NO: 56) CARGGATCAAGTCAAGGGCA ATGTAGCTGGACAGGCTTG 347 TGT G (SEQ ID NO: 57)(SEQ ID NO: 58)

Amplification was carried out using the following condition: 1 cycle of98° C. for 2 minutes for initial denaturation; 34 cycles of 98° C. for15 seconds, 62° C. for 30 seconds, and 72° C. for 45 seconds; 1 cycle of72° C. for 5 minutes; and hold at 4° C. PCR products were digested withCel-1 nuclease and resolved on a 10% acrylamide gel. Targeted mutationrates were measured using ImageJ and expressed as percent insertionsand/or deletions (% Indel). Results are summarized in Table 2. Theseresults demonstrate that both Cas9 orthologs were able to editendogenous genomic sites in human cells using a 5′-NNNNCAAA-3′ PAM(BsmCas9) or a 5′-NGAAA-3′ PAM (LrhCas9).

TABLE 2 Gene Editing with BsmCas9 and LrhCas9 in Human K562 Cells. IndelCas9 Locus/Target # Protospacer sequence (5′-3′) PAM (5′-3′) (%) BsmCas9PTN2 CTCATACATGGCTATAATAGAA GGAGCAAA 11.9 (SEQ ID NO: 59) EMX1/Target 1GAAGGTGTGGTTCCAGAACCGG AGGACAAA 6.8 (SEQ ID NO: 60) EMX1/Target 2TGGTTCCAGAACCAGGAGGACAA AGTACAAA 10.9 (SEQ ID NO: 61) EMX1/Target 3CCCAGGTGAAGGTGTGGTTCCA GAACCGGA 0 (SEQ ID NO: 62) EMX1/Target 4AGAACCGGAGGACAAAGTACAA ACGGCAGA 0 (SEQ ID NO: 63) LrhCas9 PD1L1CCTCTGGCACATCCTCCAAA TGAAA 38.9 (SEQ ID NO: 64) AAVS1CTAGGGACAGGATTGGTGAC AGAAA 32.78 (SEQ ID NO: 65) POR/Target 1GCTCGTACTGGCGAATGCT GGAAA 26.7 (SEQ ID NO: 66) POR/Target 2GCTGAAGAGCTACGAGAACC AGAAG 0 (SEQ ID NO: 67) POR/Target 3CATGGGGGAGATGGGCCGGC TGAAG 0 (SEQ ID NO: 68) CAR/Target 1AGAGACTCTCTAGAAGGGAC AGAAA 31.7 (SEQ ID NO: 69) CAR/Target 2GTGAGAGTCTCCTCCCCAATG GGAAA 27.0 (SEQ ID NO: 70) CAR/Target 3GGGAGGAGACTCTCACCTGA AGAAA 0 (SEQ ID NO: 71) *The determinantnucleotides of the PAM are underlined.

Example 3: Improvement of Parasutterella Excrementihominis Cas9(PexCas9) by Fusion with Chromatin Modulating Motifs

Parasutterella excrementihominis Cas9 (PexCas9-NLS) (SEQ ID NO:6) wasmodified by fusion with a human HMGN1 peptide (SEQ ID NO:72) on the Nterminus using a TGSG linker (SEQ ID NO:109) and with either a humanHMGB1 box A peptide (PexCas9-HN1HB1 fusion; SEQ ID NO:117) or a humanhistone H1 central globular domain peptide (PexCas9-HN1H1G; SEQ IDNO:118) on the C terminus using a LEGGGS linker (SEQ ID NO:108).

TABLE 3 Chromatin Modulating Motifs Name Peptide Sequence SEQ ID NO:HMGN1 (HN1) MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKV 72EAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAE VANQETKEDLPAENGETKTEESPASDEAGEKE AKSDHuman HMGB1 box A MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHP 73 (HB1)DASVNFSEFSKKCSERWKTMSAKEKGKFEDMA KADKARYEREMKTYIPPKGE Human histone H1STDHPKYSDMIVAAIQAEKNRAGSSRQSIQKY 74 central globular domainIKSHYKVGENADSQIKLSIKRLVTTGVLKQTK (H1G) GVGASGSFRLAKSDEP

Human K562 cells (1×10⁶) were transfected with plasmid DNA encodingPexCas9-NLS, PexCas9-HN1HB1 fusion, or PexCas9-HN1H1G fusion in molarequivalent amounts (5 and 5.4 μg, respectively) and 3 μg of sgRNAplasmid for targeting a genomic site in the human cytochrome p450oxidoreductase (POR) locus. Genomic DNA was prepared using DNAextraction solution (QuickExtract™) three days post transfection and thetargeted genomic region was PCR amplified using the forward primer5′-CTCCCCTGCTTCTTGTCGTAT-3′ (SEQ ID NO:55) and the reverse primer5′-ACAGGTCGTGGACACTCACA-3′ (SEQ ID NO:56). Amplification was carried outwith the following condition: 1 cycle of 98° C. for 2 minutes forinitial denaturation; 34 cycles of 98° C. for 15 seconds, 62° C. for 30seconds, and 72° C. for 45 seconds; 1 cycle of 72° C. for 5 minutes; andhold at 4° C. PCR products were digested with Cel-1 nuclease andresolved on a 10% acrylamide gel. Targeted mutation rates were measuredusing ImageJ and expressed as percent insertions and/or deletions (%Indel). Results are summarized in Table 4. The results demonstrate thatCas9 fusion with at least one chromatin modulating motif enhances itsgene editing efficiency on endogenous targets in human cells.

TABLE 4 Gene editing using PexCas9 and PexCas9fusion proteins in human K562 cells. Indel Cas9 nuclease LocusProtospacer (5′-3′) PAM (5′-3′)* (%) PexCas9-NLS PORTGTACATGGGGGAGATGGGC CGG 22.9 PexCas9-HN1HB1 fusion (SEQ ID NO: 75) 36.8PexCas9HN1H1G fusion 43.7 *The determinant nucleotides of the PAM areunderlined.

Example 4. Improvement of Mycoplasma canis Cas9 (McaCas9) System bysgRNA Modification

The wild type crRNA coding sequence of McaCas9 contains four consecutivethymidine residues in the repeat region, and three of the four thymidineresidues are predicted to pair with three adenosine residues in theputative tracrRNA sequence when the crRNA and tracrRNA are joinedtogether to form a sgRNA. Human RNA polymerase (Pol) III is known to usefour or more consecutive thymidine residues on the coding RNA strand asa transcription termination signal. To prevent an early transcriptionaltermination of McaCas9 sgRNA in human cells, a T to C mutation and acorresponding A to G mutation were introduced into the sgRNA scaffold toform a modified sgRNA scaffold with the following sequence:5′-GUUCUAGUGUUGUACAAUAUUUGGGUGAAAACCCAAAUAUUGUACAUCCUAGAUCAAGGCGCUUAAUUGCUGCCGUAAUUGCUGAAAGCGUAGCUUUCAGUUUUUUU-3′ (SEQ ID NO:76),where the mutated nucleotides are underlined. This modification is alsopredicted to increase the sgRNA scaffold thermodynamic stability.

Human K562 cells (1×10⁶) were transfected with 5.5 μg of plasmid DNAencoding a McaCas9 fusion protein, which contains a HMGN1 peptide on theN terminus and a histone H1 globular domain peptide on the C terminus,and 3 μg of sgRNA plasmid DNA encoding the control sgRNA scaffold or themodified sgRNA scaffold. Genomic DNA was prepared using a DNA extractionsolution (QuickExtract™) three days post transfection and the targetedgenomic region was PCR amplified using the forward primer5′-CTCCCCTGCTTCTTGTCGTAT-3′ (SEQ ID NO:55) and the reverse primer5′-ACAGGTCGTGGACACTCACA-3′ (SEQ ID NO:56). Amplification was carried outwith the following condition: 1 cycle of 98° C. for 2 minutes forinitial denaturation; 34 cycles of 98° C. for 15 seconds, 62° C. for 30seconds, and 72° C. for 45 seconds; 1 cycle of 72° C. for 5 minutes; andhold at 4° C. PCR products were digested with Cel-1 nuclease andresolved on a 10% acrylamide gel. Targeted mutation rates were measuredusing ImageJ and expressed as percent insertions and/or deletions (%Indel). Results are summarized in Table 5. The results demonstrate thatthe activity of a Cas9 ortholog in mammalian cells can be enhanced bymodifying its sgRNA scaffold.

TABLE 5 Gene editing using McaCas9 in combination withcontrol sgRNA scaffold or modified sgRNA scaffold. Indel sgRNA scaffoldLocus Protospacer (5′-3′) PAM (5′-3′) (%) Control sgRNA PORATAGATGCGGCCAAGGTGTACA TGGG 25.5 scaffold (SEQ ID NO: 77) Modified sgRNA36.2 scaffold *The determinant nucleotides of the PAM are underlined.

Example 5. Improvement of McaCas9, BsmCas9, PexCas9, and LrhCas9Activity by Fusion with Chromatin Modulating Motifs

Additional Cas9-CMM fusion proteins were prepared by linkingMcaCas9-NLS, BsmCas9-NLS, and LrhCas9-NLS proteins with HMGN1 (HN1) atthe amino terminus and either HMGB1 box A (HB1) or histone H1 centralglobular motif (H1 G) at the carboxyl terminus to yield McaCas9-HN1HB1(SEQ ID NO:123), McaCas9-HN1H1G (SEQ ID NO:124), BsmCas9-HN1HB1 (SEQ IDNO:119), Bsm-HN1H1G (SEQ ID NO:120), Lrh-HN1HB1 (SEQ ID NO:121),LrhCas9-HN1H1G (SEQ ID NO:122). The nuclease activity of these fusionsand the PexCas9-CMM fusions described above in Example 3 were comparedto the activity of the corresponding engineered Cas9 protein essentiallyas described above in Examples 2 and 3. Table 6 presents the target site(i.e., protospacer+PAM, which is shown in bold with the determinatenucleotides underlined) in specific loci for each Cas9 nuclease.

TABLE 6 Gene Editing Target Sites SEQ Cas9 Locus Target site (5′-3′)ID NO Mca PORI1 ATAGATGCGGCCAAGGTGTACATG GG 125 POR2CTACGAGAACCAGAAGCCGTGAGT GG 126 Bsm PTN2 CTCATACATGGCTATAATAGAAGGAG CAAA127 EMX1 GAAGGTGTGGTTCCAGAACCGGAGGA CAAA 128 EMX2TGGTTCCAGAACCGGAGGACAAAGTA CAAA 129 Pex POR1 TGTACATGGGGGAGATGGGCC GG130 AAVS1 GGGGCCACTAGGGACAGGATT GG 131 Lrh PORI1 AGCTCGTACTGGCGAATGCTGGAAA 132 PD1L1 CCTCTGGCACATCCTCCAAAT GAAA 133

The percent of indels under each condition are plotted in FIGS. 2A-D.Both the HN1HB1 and HN1H1G combinations significantly enhanced the fourCas9 orthologs on at least one site. Based on the fold change magnitude,the CMM fusion modification provided the largest enhancement on McaCas9,increasing its activity by at least five-fold on the two sites tested(FIG. 2A). CMM fusion provided more than two-fold enhancement on PexCas9(FIG. 2B). BsmCas9 activity was enhanced by more than three-fold on onesite, but there was only 20% increase on the second site and no effecton the third site (FIG. 2C). It should be noted, however, that all threeBsmCas9 nucleases were highly efficient (>35% indels). LrhCas9 washighly efficient on the two sites tested (22% and 33% indels) evenwithout the fusion modification (FIG. 2D). However, the HN1H1Gcombination still provided a significant enhancement on both sites, with70% and 28% increase in activity. These results demonstrate that the CMMfusion strategy enhances gene editing efficiency.

Example 6. Off-Target Effects of Cas9-CMM Fusions

To assess the off-target activity of the Cas9-CMM fusions, 1 to 5 topranked potential off-target sites for each target site were analyzedusing the Surveyor Nuclease assay. In addition to the Cas9 and Cas9-CMMfusion data described above in Example 5, data from Streptococcuspyogenes Cas9 (SpyCas9), SpyCas9-CMM fusions, Streptococcus pasteurianusCas9 (SpaCas9), Spa-CMM fusions, Campylobacter jejuni Cas9 (CjeCas9),and CjeCas9-SMM fusions were also analyzed. From a total of 64 potentialoff-target sites assayed, off-target cleavage was detected on 11 sites,contributed by 9 guide sequences out of total 21 guide sequences tested.On the 11 off-target sites, the control Cas9 and the fusion nucleaseswere concurrent, with the exception of the POR Spy 1-OT1 site, where nooff-target cleavage was detected on the control SpyCas9. Overall, therewas no significant difference between the fusion nucleases and thecontrol Cas9 (FIG. 3). For example, across all the 11 off-target sites,the HN1H1G fusion combination averaged 8.0±6.0% indels and the controlCas9 averaged 7.5±5.1% indels. Likewise, across the 10 off-target sitesrelevant to the HN1HB1 fusion combination, there was no significantdifference between the fusion combination and the control Cas9 (6.9±5.7%vs. 6.5±5.4% indels). Taken together, these results show that theon-target activity enhancement by the HN1H1B and HN1H1G fusioncombinations generally does not result in an increase in off-targetactivity.

Engineered Cas9 Systems

Table 7 presents the human codon optimized DNA and protein sequences ofengineered Cas9/NLS proteins (SEQ ID NOS:1-30, wherein the NLS sequenceis underlined) and the DNA sequences of engineered sgRNAs (SEQ IDNOS:31-45; the N residues at the 5′ end indicate the programmable targetsequence). Also presented are the Cas9-CMM fusions (SEQ ID NOS:117-124).

TABLE 7 Engineered Cas9 Systems BsmCas9/NLS DNA sequence (SEQ ID NO: 1)ATGAACTACAAGATGGGCCTCGACATCGGAATCGCCTCTGTTGGATGGGCCGTGATCAACCTGGACCTGAAGAGAATCGAGGACCTCGGCGTGCGGATCTTCGACAAGGCTGAGCATCCTCAGAACGGCGAGTCTCTGGCCCTGCCTAGAAGAATTGCCAGAAGCGCCAGACGGCGGCTGCGGAGAAGAAAGCACAGACTGGAACGGATCAGACGGCTGCTGGTGTCCGAGAACGTGCTGACCAAAGAAGAGATGAACCTGCTGTTCAAGCAGAAAAAGCAGATCGACGTGTGGCAGCTGAGAGTGGACGCCCTGGAAAGAAAGCTGAACAACGACGAGCTGGCCAGAGTGCTGCTGCACCTGGCCAAGAGAAGAGGCTTCAAGAGCAACAGAAAGAGCGAGCGGAACAGCAAAGAGAGCAGCGAGTTCCTGAAGAACATCGAAGAGAACCAGAGCATTCTGGCCCAGTACAGATCCGTGGGCGAGATGATCGTGAAGGACAGCAAGTTCGCCTACCACAAGCGGAACAAGCTGGACAGCTACAGCAACATGATCGCCAGGGACGATCTGGAAAGAGAGATCAAGCTGATCTTCGAGAAGCAGCGCGAGTTCAACAACCCCGTGTGCACCGAGAGACTGGAAGAGAAGTACCTGAACATCTGGTCCAGCCAGCGGCCTTTCGCCTCCAAAGAGGACATCGAGAAAAAAGTGGGCTTCTGCACCTTCGAGCCCAAAGAGAAAAGAGCCCCTAAGGCCACCTACACCTTCCAGAGCTTCATCGTGTGGGAGCACATCAACAAGCTGCGGCTGGTGTCTCCCGACGAGACAAGAGCCCTGACCGAGATCGAGCGGAATCTGCTGTATAAGCAGGCCTTCAGCAAGAACAAGATGACCTACTACGACATCCGGAAGCTGCTGAACCTGAGCGACGACATCCACTTCAAGGGCCTGCTGTACGACCCCAAGAGCAGCCTGAAGCAGATTGAGAACATCCGGTTTCTGGAACTGGACTCTTACCACAAGATCCGGAAGTGCATCGAGAATGTGTACGGCAAGGACGGCATCCGCATGTTCAACGAGACAGACATCGACACCTTCGGCTACGCCCTGACCATCTTCAAGGACGACGAGGATATCGTGGCCTACCTGCAGAACGAGTACATCACCAAGAACGGCAAGCGGGTGTCCAATCTGGCCAACAAGGTGTACGACAAGTCCCTGATCGACGAACTGCTGAATCTGTCCTTCTCCAAATTCGCCCACCTGAGCATGAAGGCCATCCGGAACATCCTGCCTTACATGGAACAGGGCGAAATCTACAGCAAGGCCTGCGAACTGGCCGGCTACAACTTCACAGGCCCCAAGAAGAAAGAGAAGGCCCTGCTGCTGCCTGTGATCCCCAATATCGCCAATCCTGTGGTCATGCGGGCCCTGACACAGAGCAGAAAGGTGGTCAACGCCATCATCAAGAAATACGGATCCCCCGTGTCCATCCACATCGAGCTGGCTAGGGATCTGAGCCACAGCTTCGACGAGCGGAAGAAGATCCAGAAGGACCAGACCGAGAACCGCAAGAAGAACGAAACCGCCATCAAGCAGCTGATCGAGTACGAGCTGACTAAGAACCCCACCGGCCTGGACATCGTGAAGTTCAAACTTTGGAGCGAGCAGCAAGGCAGATGCATGTACTCCCTGAAGCCTATTGAGCTGGAAAGACTGCTGGAACCCGGCTACGTGGAAGTGGACCACATTCTGCCCTACAGCAGAAGCCTGGACGACAGCTACGCCAACAAAGTGCTGGTCCTGACAAAAGAGAACCGCGAAAAGGGCAATCACACCCCTGTGGAATATCTCGGCCTGGGCTCTGAGCGGTGGAAGAAATTCGAGAAGTTCGTGCTGGCTAACAAGCAGTTCTCTAAGAAGAAGAAGCAGAACCTGCTCCGGCTGAGATACGAGGAAACCGAGGAAAAAGAGTTCAAAGAGCGGAACCTGAACGACACCCGGTACATCTCCAAGTTCTTCGCCAACTTCATCAAAGAGCATCTGAAGTTCGCCGACGGCGACGGCGGCCAGAAAGTGTACACAATCAACGGCAAGATCACCGCTCACCTGAGAAGCAGATGGGACTTCAACAAGAACCGGGAAGAGAGCGACCTGCACCACGCTGTGGATGCTGTGATTGTGGCCTGTGCCACACAGGGCATGATCAAGAAGATTACCGAGTTCTACAAGGCCCGCGAGCAGAACAAAGAGTCCGCCAAGAAAAAAGAACCCATCTTTCCCCAGCCTTGGCCTCACTTCGCCGATGAGCTGAAGGCTCGGCTGAGCAAGTTCCCTCAAGAGTCCATCGAGGCCTTCGCTCTGGGCAACTACGACAGAAAGAAGCTGGAATCCCTGCGGCCTGTGTTCGTGTCCAGAATGCCCAAGAGATCCGTGACAGGCGCTGCCCACCAAGAGACACTGAGAAGATGCGTGGGCATCGACGAGCAGTCTGGCAAGATTCAGACCGCCGTGAAAACAAAGCTGAGCGACATCAAGCTGGATAAGGACGGACACTTCCCCATGTACCAGAAAGAGTCTGACCCCAGAACCTACGAGGCCATCAGACAGAGGCTGCTCGAACACAACAACGACCCTAAGAAGGCCTTTCAAGAGCCACTGTACAAGCCCAAAAAGAATGGCGAGCCCGGACCAGTGATCCGGACCGTGAAGATCATCGACACAAAGAACAAGGTGGTGCACCTGGACGGCAGCAAGACAGTGGCCTACAACTCCAACATCGTGCGGACCGACGTGTTCGAGAAGGATGGCAAGTACTACTGCGTGCCCGTGTACACTATGGATATCATGAAGGGCACCCTGCCTAACAAGGCCATCGAAGCCAACAAGCCCTACTCCGAGTGGAAAGAGATGACCGAAGAGTACACGTTCCAGTTCAGTCTGTTCCCCAACGACCTCGTGCGCATCGTGCTGCCAAGAGAGAAAACCATCAAGACCAGCACCAACGAGGAAATCATCATTAAGGACATCTTTGCCTACTACAAGACCATCGACAGCGCCACAGGCGGCCTGGAACTGATCTCCCACGATCGGAACTTCAGCCTGAGAGGCGTGGGCTCTAAGACACTGAAGCGCTTTGAGAAGTATCAGGTGGACGTGCTGGGCAACATCCACAAAGTGAAGGGCGAGAAGAGAGTCGGCCTGGCCGCTCCTACCAACCAGAAAAAGGGAAAGACCGTGGACAGCCTGCAGAGCGTGTCCGATCCCAAGAAGAAGAGGAAGGTG BsmCas9/NLS protein sequence (SEQ ID NO: 2)MNYKMGLDIGIASVGWAVINLDLKRIEDLGVRIFDKAEHPQNGESLALPRRIARSARRRLRRRKHRLERIRRLLVSENVLTKEEMNLLFKQKKQIDVWQLRVDALERKLNNDELARVLLHLAKRRGFKSNRKSERNSKESSEFLKNIEENQSILAQYRSVGEMIVKDSKFAYHKRNKLDSYSNMIARDDLEREIKLIFEKQREFNNPVCTERLEEKYLNIWSSQRPFASKEDIEKKVGFCTFEPKEKRAPKATYTFQSFIVWEHINKLRLVSPDETRALTEIERNLLYKQAFSKNKMTYYDIRKLLNLSDDIHFKGLLYDPKSSLKQIENIRFLELDSYHKIRKCIENVYGKDGIRMFNETDIDTFGYALTIFKDDEDIVAYLQNEYITKNGKRVSNLANKVYDKSLIDELLNLSFSKFAHLSMKAIRNILPYMEQGEIYSKACELAGYNFTGPKKKEKALLLPVIPNIANPVVMRALTQSRKVVNAIIKKYGSPVSIHIELARDLSHSFDERKKIQKDQTENRKKNETAIKQLIEYELTKNPTGLDIVKFKLWSEQQGRCMYSLKPIELERLLEPGYVEVDHILPYSRSLDDSYANKVLVLTKENREKGNHTPVEYLGLGSERWKKFEKPVLANKQFSKKKKQNLLRLRYEETEEKEFKERNLNDTRYISKFFANFIKEHLKFADGDGGQKVYTINGKITAHLRSRWDFNKNREESDLHHAVDAVIVACATQGMIKKITEFYKAREQNKESAKKKEPIFPQPWPHFADELKARLSKFPQESIEAFALGNYDRKKLESLRPVFVSRMPKRSVTGAAHQETLRRCVGIDEQSGKIQTAVKTKLSDIKLDKDGHFPMYQKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNKVVHLDGSKTVAYNSNIVRTDVFEKDGKYYCVPVYTMDIMKGTLPNKAIEANKPYSEWKEMTEEYTFQFSLFPNDLVRIVLPREKTIKTSTNEEIIIKDIFAYYKTIDSATGGLELISHDRNFSLRGVGSKTLKRFEKYQVDVLGNIHKVKGEKRVGLAAPTNQKKGKTVDSLQSVSDPKKKRKV LrhCas9/NLS DNA sequence (SEQ ID NO: 3)ATGACCAAGCTGAACCAGCCTTACGGCATCGGCCTGGACATCGGCAGCAATAGCATCGGCTTTGCCGTGGTGGACGCCAACAGCCATCTGCTGAGACTGAAGGGCGAGACAGCCATCGGCGCCAGACTGTTTAGAGAGGGACAGAGCGCCGCTGACAGACGGGGAAGCAGAACCACAAGAAGGCGGCTGTCCAGAACCAGATGGCGGCTGAGCTTCCTGCGGGATTTCTTCGCCCCTCACATCACCAAGATCGACCCCGACTTCTTTCTGCGGCAAAAATACTCCGAGATCAGCCCCAAGGACAAGGACAGGTTTAAGTACGAGAAGCGGCTGTTCAACGACCGGACCGACGCCGAGTTCTACGAGGACTACCCCAGCATGTACCACCTGAGACTGCACCTGATGACCCACACACACAAGGCCGATCCTCGGGAAATCTTCCTGGCCATCCACCACATCCTGAAGTCCAGAGGCCACTTTCTGACACCCGGCGCTGCCAAGGACTTCAACACCGACAAAGTGGACCTTGAGGACATCTTCCCCGCTCTGACAGAGGCTTACGCCCAGGTGTACCCCGATCTGGAACTGACCTTCGATCTGGCCAAGGCCGACGACTTCAAGGCCAAGCTGCTGGACGAACAGGCCACACCTAGCGACACACAGAAAGCCCTGGTCAACCTGCTGCTGTCTAGCGACGGCGAGAAAGAAATCGTGAAGAAGCGGAAGCAGGTCCTGACCGAGTTCGCCAAGGCCATCACCGGCCTGAAAACAAAGTTCAATCTGGCCCTGGGCACCGAGGTGGACGAAGCTGATGCTTCCAACTGGCAGTTCAGCATGGGCCAGCTGGACGACAAGTGGTCCAACATCGAAACCAGCATGACCGACCAGGGCACCGAAATCTTCGAGCAGATCCAAGAGCTGTACCGGGCCAGACTGCTGAACGGAATTGTGCCTGCCGGCATGAGCCTGTCTCAGGCCAAAGTGGCCGATTACGGCCAGCACAAAGAGGACCTGGAACTGTTCAAGACCTACCTGAAGAAGCTGAACGACCACGAGCTGGCCAAGACCATCAGGGGCCTGTACGATCGGTACATCAACGGCGACGACGCCAAGCCTTTCCTGCGCGAGGATTTTGTGAAGGCCCTGACCAAAGAAGTGACAGCTCACCCCAACGAGGTGTCCGAACAGCTGCTGAACAGGATGGGCCAAGCCAACTTCATGCTGAAGCAGCGGACCAAGGCCAACGGCGCCATTCCTATTCAGCTGCAGCAGAGAGAGCTGGACCAGATCATTGCCAACCAGAGCAAGTACTACGACTGGCTGGCCGCTCCTAATCCTGTGGAAGCCCACAGATGGAAGATGCCCTACCAGCTGGATGAGCTGCTCAACTTTCACATCCCCTACTACGTGGGCCCTCTGATCACCCCTAAACAGCAGGCCGAGAGCGGCGAGAATGTGTTCGCTTGGATGGTCCGAAAGGACCCCAGCGGCAACATCACCCCTTACAACTTCGACGAGAAGGTGGACAGAGAGGCCAGCGCCAACACCTTCATCCAGAGAATGAAGACCACCGACACATACCTGATCGGCGAGGACGTGCTGCCTAAGCAGAGCCTGCTGTACCAGAAATACGAGGTGCTGAACGAGCTGAACAACGTGCGGATCAACAACGAGTGCCTGGGCACAGACCAGAAGCAGAGACTGATCAGAGAGGTGTTCGAGCGGCACAGCAGCGTGACCATCAAACAGGTGGCCGACAATCTGGTGGCCCACGGCGATTTTGCCAGACGGCCTGAGATTAGAGGACTGGCCGATGAGAAGCGGTTCCTGAGCAGCCTGAGCACCTACCACCAGCTGAAAGAGATCCTGCACGAGGCCATCGACGACCCCACCAAACTGCTGGATATCGAGAACATCATCACCTGGTCCACCGTGTTCGAGGACCACACCATCTTCGAGACAAAGCTGGCCGAGATCGAGTGGCTGGACCCCAAGAAGATCAACGAGCTGTCTGGCATCAGATACAGAGGCTGGGGCCAGTTCTCCCGGAAGCTGCTCGATGGACTGAAGCTTGGCAATGGCCACACCGTGATTCAAGAACTGATGCTGAGCAACCACAACCTGATGCAGATCCTGGCCGACGAGACACTGAAAGAAACCATGACAGAGCTGAATCAGGACAAGCTGAAAACCGACGACATCGAGGATGTGATCAACGACGCCTACACAAGCCCCAGCAACAAAAAGGCCCTCAGACAGGTGCTGAGAGTGGTCGAGGATATCAAGCACGCCGCCAACGGACAGGACCCTAGCTGGCTGTTTATCGAAACCGCCGATGGAACAGGCACCGCCGGCAAGAGAACACAGAGCCGGCAGAAACAGATCCAGACCGTGTACGCCAACGCCGCTCAAGAGCTGATCGATTCTGCCGTGCGGGGCGAGCTGGAAGATAAGATTGCTGACAAGGCCAGCTTCACCGACCGGCTGGTGCTGTACTTTATGCAAGGCGGCAGAGACATCTACACAGGCGCCCCTCTGAACATCGACCAGCTGAGCCACTACGATATCGACCACATTCTGCCCCAGAGCCTGATCAAGGACGACAGCCTGGACAACCGGGTGCTCGTGAACGCCACCATCAACCGCGAGAAGAACAATGTGTTTGCCAGCACACTGTTCGCCGGAAAGATGAAGGCCACCTGGCGGAAATGGCACGAAGCCGGACTGATCTCTGGCAGAAAGCTGCGGAATCTGATGCTGCGGCCCGACGAGATCGACAAGTTTGCCAAGGGCTTCGTGGCCCGGCAGCTGGTTGAGACAAGACAGATCATCAAGCTGACAGAGCAGATTGCCGCCGCTCAGTACCCCAACACCAAGATTATTGCCGTGAAGGCCGGACTGTCCCATCAGCTGAGAGAGGAACTGGACTTCCCCAAGAACCGGGACGTGAACCACTACCACCACGCCTTCGATGCCTTTCTGGCCGCTAGAATCGGCACCTACCTGCTGAAGAGATACCCCAAGCTGGCCCCATTCTTCACCTACGGCGAGTTTGCTAAGGTGGACGTCAAGAAGTTCCGCGAGTTCAACTTCATCGGAGCCCTGACACACGCCAAGAAGAACATTATCGCCAAGGACACCGGCGAGATCGTGTGGGACAAAGAGCGGGACATCAGAGAACTGGACCGCATCTACAACTTCAAGCGGATGCTGATCACACACGAGGTGTACTTCGAGACTGCCGACCTGTTCAAGCAGACCATCTACGCCGCTAAGGACAGCAAAGAGAGAGGCGGCAGCAAGCAGCTGATCCCTAAGAAGCAGGGCTACCCCACTCAGGTGTACGGCGGCTACACACAAGAGAGCGGCTCTTACAACGCCCTCGTCAGAGTGGCCGAGGCCGATACAACAGCCTACCAAGTGATCAAGATCAGCGCCCAGAACGCCAGCAAGATCGCCTCCGCCAACCTGAAAAGCCGCGAGAAAGGCAAACAGCTCCTGAATGAGATCGTCGTGAAGCAGCTGGCTAAGCGGCGGAAGAACTGGAAGCCTAGCGCCAATAGCTTCAAGATCGTGATCCCCAGATTCGGCATGGGCACCCTGTTCCAGAACGCTAAGTACGGCCTGTTCATGGTCAACAGCGACACCTACTACCGGAACTACCAAGAACTCTGGCTGAGCCGGGAAAACCAGAAACTGCTGAAAAAGCTGTTCTCCATCAAATACGAGAAAACCCAGATGAACCACGACGCCCTGCAGGTCTACAAGGCCATTATCGACCAGGTGGAAAAGTTCTTCAAGCTGTACGACATCAACCAGTTCCGCGCCAAGCTGAGCGACGCCATCGAGAGATTTGAGAAGCTGCCCATCAATACCGACGGCAACAAGATCGGCAAGACCGAGACTCTGAGACAGATCCTGATCGGACTGCAGGCCAATGGCACCCGGTCCAACGTGAAGAACCTGGGCATCAAGACCGATCTGGGCCTGCTGCAAGTCGGCAGCGGAATCAAGCTGGACAAGGATACCCAGATCGTGTATCAGAGCCCCTCCGGCCTGTTTAAGCGGAGAATCCCACTGGCTGACCTGCCCAAGAAGAAGAGGAAGGTG LrhCas9/NLS protein sequence (SEQ ID NO: 4)MTKLNQPYGIGLDIGSNSIGFAVVDANSHLLRLKGETAIGARLFREGQSAADRRGSRTTRRRLSRTRWRLSFLRDFFAPHITKIDPDFFLRQKYSEISPKDKDRFKYEKRLFNDRTDAEFYEDYPSMYHLRLHLMTHTHKADPREIFLAIHHILKSRGHFLTPGAAKDFNTDKVDLEDIFPALTEAYAQVYPDLELTFDLAKADDFKAKLLDEQATPSDTQKALVNLLLSSDGEKEIVKKRKQVLTEFAKAITGLKTKFNLALGTEVDEADASNWQFSMGQLDDKWSNIETSMTDQGTEIFEQIQELYRARLLNGIVPAGMSLSQAKVADYGQHKEDLELFKTYLKKLNDHELAKTIRGLYDRYINGDDAKPFLREDFVKALTKEVTAHPNEVSEQLLNRMGQANFMLKQRTKANGAIPIQLQQRELDQIIANQSKYYDWLAAPNPVEAHRWKMPYQLDELLNFHIPYYVGPLITPKQQAESGENVFAWMVRKDPSGNITPYNFDEKVDREASANTFIQRMKTITTYLIGEDVLPKQSLLYQKYEVLNELNNVRINNECLGTDQKQRLIREVFERHSSVTIKQVADNLVAHGDFARRPEIRGLADEKRFLSSLSTYHQLKEILHEAIDDPTKLLDIENIITWSTVFEDHTIFETKLAEIEWLDPKKINELSGIRYRGWGQFSRKLLDGLKLGNGHTVIQELMLSNHNLMQILADETLKETMTELNQDKLKTDDIEDVINDAYTSPSNKKALRQVLRVVEDIKHAANGQDPSWLFIETADGIGTAGKRTQSRQKQIQTVYANAAQELIDSAVRGELEDKIADKASFTDRLVLYFMQGGRDIYTGAPLNIDQLSHYDIDHILPQSLIKDDSLDNRVLVNATINREKNNVFASTLFAGKMKATWRKWHEAGLISGRKLRNLMLRPDEIDKFAKGFVARQLVETRQIIKLTEQIAAAQYPNTKIIAVKAGLSHQLREELDFPKNRDVNHYHHAFDAFLAARIGTYLLKRYPKLAPFFTYGEFAKVDVKKFREFNFIGALTHAKKNITAKDTGEIVWDKERDIRELDRIYNFKRMLITHEVYFETADLFKQTIYAAKDSKERGGSKQLIPKKQGYPTQVYGGYTQESGSYNALVRVAEADTTAYQVIKISAQNASKIASANLKSREKGKQLLNEIVVKQLAKRRKNWKPSANSFKIVIPRFGMGTLFQNAKYGLFMVNSDTYYRNYQELWLSRENQKLLKKLFSIKYEKTQMNHDALQVYKAIIDQVEKFFKLYDINQFRAKLSDAIERFEKLPINTDGNKIGKTETLRQILIGLQANGTRSNVKNLGIKTDLGLLQVGSGIKLDKDTQIVYQSPSGLFKRRIPLADLPKKKRKVPexCas9/NLS DNA sequence (SEQ ID NO: 5)ATGGGCAAGACCCACATCATCGGCGTTGGCCTGGATCTCGGCGGCACATACACAGGCACCTTCATCACCAGCCATCCTAGCGACGAAGCCGAGCACAGAGATCACAGCAGCGCCTTCACCGTGGTCAACAGCGAGAAGCTGAGCTTCAGCAGCAAGAGCAGAACAGCCGTGCGGCACAGAGTGCGGAGCTACAAGGGCTTCGACCTGCGTAGAAGGCTGCTGCTTCTGGTGGCCGAGTATCAGCTGCTGCAGAAGAAGCAGACACTGGCCCCTGAGGAAAGAGAGAACCTGAGAATCGCCCTGAGCGGCTACCTGAAGAGAAGAGGCTACGCCAGAACCGAGGCCGAGACAGATACAAGCGTGCTGGAATCTCTGGACCCCAGCGTGTTCAGCAGCGCTCCCAGCTTCACCAATTTCTTCAACGACAGCGAGCCCCTGAACATCCAGTGGGAAGCCATTGCCAACTCTCCCGAGACAACAAAGGCCCTGAACAAAGAGCTGAGCGGCCAGAAAGAGGCCGACTTCAAGAAGTACATCAAGACCAGCTTTCCCGAGTACAGCGCCAAAGAGATTCTGGCCAACTACGTGGAAGGCAGACGGGCCATTCTGGACGCCAGCAAGTATATCGCCAACCTGCAGAGCCTGGGCCACAAGCACAGAAGCAAGTACCTGAGCGACATTCTGCAGGACATGAAGCGGGACAGCCGGATCACCAGACTGAGCGAAGCCTTTGGCAGCACCGACAACCTGTGGCGGATCATCGGCAACATCAGCAATCTGCAAGAACGGGCCGTGCGGTGGTACTTCAACGATGCCAAGTTCGAGCAGGGCCAAGAGCAGCTGGATGCCGTGAAGCTGAAGAATGTGCTCGTGCGGGCCCTGAAGTATCTGCGGAGTGACGACAAAGAGTGGAGCGCCTCTCAGAAGCAGATCATCCAGTCTCTGGAACAGAGCGGCGACGTGCTGGATGTGCTGGCTGGACTCGACCCCGACAGAACAATCCCTCCATACGAGGACCAGAACAACAGACGGCCTCCTGAGGATCAGACCCTGTATCTGAACCCCAAGGCTCTGAGCAGCGAGTACGGCGAGAAGTGGAAGTCTTGGGCCAACAAGTTTGCCGGCGCTTACCCTCTGCTGACCGAGGATCTGACCGAGATCCTGAAGAACACCGACAGAAAGTCCCGGATCAAGATCAGATCCGATGTGCTGCCCGACAGCGACTACAGACTGGCCTACATCCTGCAGAGAGCCTTCGATCGGTCTATCGCCCTGGACGAGTGCAGCATCAGAAGAACCGCCGAGGACTTCGAGAACGGCGTGGTCATCAAGAACGAGAAACTGGAAGATGTGCTGAGCGGACACCAGCTGGAAGAGTTTCTGGAATTTGCCAATCGGTACTACCAAGAGACAGCCAAGGCCAAGAACGGCCTGTGGTTCCCAGAGAACGCCCTGCTGGAAAGAGCCGATCTGCACCCTCCTATGAAGAACAAGATTCTGAACGTGATCGTCGGACAGGCCCTGGGAGTGTCTCCTGCTGAGGGCACCGATTTCATCGAGGAAATTTGGAACAGCAAAGTGAAAGGCCGGTCCACCGTGCGGAGCATCTGTAACGCCATCGAGAATGAGAGAAAGACCTACGGACCCTACTTCAGCGAGGACTACAAGTTCGTGAAAACGGCCCTGAAAGAGGGCAAAACCGAGAAAGAGCTGTCCAAGAAATTCGCCGCCGTGATCAAGGTGCTGAAGATGGTGTCTGAGGTGGTGCCCTTTATCGGAAAAGAGCTGCGGCTGTCTGACGAGGCCCAGAGCAAGTTCGACAATCTGTACTCTCTGGCCCAGCTGTACAACCTGATCGAGACAGAGCGGAACGGCTTCAGCAAGGTGTCACTGGCTGCCCACCTGGAAAATGCCTGGCGGATGACCATGACAGATGGATCCGCCCAGTGCTGTAGACTGCCTGCCGATTGTGTGCGGCCCTTCGACGGCTTTATCCGGAAGGCCATCGACCGGAACTCTTGGGAAGTCGCCAAGCGGATTGCCGAGGAAGTGAAGAAGTCCGTCGACTTCACCAACGGCACCGTGAAGATCCCTGTGGCCATCGAGGCCAACAGCTTCAACTTTACCGCCAGCCTGACCGACCTGAAGTACATTCAGCTCAAAGAACAGAAGCTCAAGAAGAAGTTGGAGGACATCCAGCGGAACGAAGAGAATCAAGAGAAGCGGTGGCTGAGCAAAGAGGAACGGATCAGAGCCGACAGCCACGGCATCTGTGCCTATACTGGCAGACCCCTGGATGACGTGGGCGAGATCGATCACATCATCCCCAGAAGCCTGACACTGAAGAAAAGCGAGAGCATCTACAACTCCGAAGTGAACCTGATCTTCGTGTCTGCCCAGGGCAATCAAGAAAAGAAGAACAACATCTACCTGCTGAGCAACCTCGCCAAGAACTACCTGGCCGCCGTGTTTGGCACAAGCGACCTGAGCCAGATCACCAACGAGATCGAGAGCACCGTGCTGCAGCTGAAAGCTGCTGGCAGACTGGGCTACTTCGATCTGCTGAGCGAAAAAGAGCGGGCCTGCGCCAGACATGCCCTGTTTCTGAATAGCGACTCCGAGGCCAGACGCGCCGTGATTGATGTTCTTGGCTCTCGGAGAAAGGCCAGCGTGAACGGAACCCAGGCTTGGTTTGTGCGGTCCATCTTCTCCAAAGTGCGGCAGGCACTGGCCGCTTGGACACAAGAAACAGGCAACGAGCTGATCTTTGACGCCATCAGCGTGCCAGCCGCCGATAGCTCTGAGATGAGGAAGAGATTCGCCGAGTACCGGCCTGAGTTCAGAAAGCCCAAAGTGCAGCCTGTGGCCTCTCACAGCATCGACGCCATGTGCATCTATCTGGCCGCCTGCAGCGACCCCTTCAAGACCAAGAGAATGGGCTCTCAGCTGGCCATCTACGAGCCCATCAACTTCGATAACCTGTTCACCGGCAGCTGTCAAGTGATCCAGAACACCCCTCGGAACTTCTCCGACAAGACCAATATCGCTAACAGCCCCATCTTCAAAGAGACAATCTACGCCGAGCGGTTCCTGGACATCATCGTGTCCAGAGGCGAGATTTTCATCGGCTACCCCAGCAACATGCCCTTCGAGGAAAAGCCCAACCGGATCAGCATCGGCGGCAAGGACCCTTTCAGCATCCTGTCTGTGCTGGGCGCCTACCTGGATAAGGCCCCTAGCAGCGAGAAAGAAAAGCTCACCATCTACCGGGTCGTCAAGAACAAAGCCTTCGAGCTGTTCTCCAAGGTGGCCGGCAGCAAGTTTACCGCCGAAGAAGATAAGGCCGCCAAGATCCTGGAAGCCCTGCACTTCGTGACCGTGAAACAGGATGTGGCCGCCACCGTGTCCGATCTGATCAAGAGCAAGAAAGAACTGAGCAAGGATAGCATCGAGAACCTGGCCAAGCAGAAGGGCTGCCTGAAGAAGGTGGAATACTCCAGCAAAGAGTTCAAGTTCAAGGGCAGCCTGATCATCCCTGCCGCCGTGGAATGGGGAAAAGTGCTGTGGAACGTGTTCAAAGAAAACACGGCCGAAGAACTGAAGGACGAGAACGCTCTGAGGAAGGCCCTGGAAGCTGCCTGGCCTAGCTCTTTCGGCACCAGAAACCTGCACTCTAAGGCCAAGCGGGTGTTCAGCCTGCCTGTGGTGGCTACACAATCTGGCGCCGTGCGGATCAGACGCAAGACAGCCTTCGGCGACTTCGTGTACCAGAGCCAGGACACAAACAACCTGTACAGCAGCTTCCCCGTGAAGAACGGCAAGCTGGATTGGAGCAGCCCTATCATTCACCCCGCTCTGCAGAACCGGAACCTGACCGCCTACGGCTACAGATTCGTGGACCACGACAGATCCATCAGCATGAGCGAGTTCAGAGAGGTGTACAACAAGGACGACCTGATGCGGATCGAGCTGGCCCAGGGAACAAGCAGCAGACGCTACCTGAGAGTGGAAATGCCCGGCGAGAAATTCCTCGCTTGGTTTGGCGAGAACAGCATCAGCCTGGGCTCCAGCTTCAAGTTCTCTGTGTCCGAGGTGTTCGACAACAAAATCTACACCGAGAACGCCGAGTTTACCAAGTTCCTGCCTAAGCCTAGAGAGGACAACAAGCACAACGGGACCATCTTTTTCGAACTCGTGGGCCCCAGAGTGATCTTCAACTACATCGTTGGCGGAGCCGCCAGCAGCCTGAAAGAAATCTTTAGCGAGGCCGGCAAAGAGCGGAGCCCCAAGAAGAAGAGGAAGGTG PexCas9/NLS protein sequence (SEQ ID NO: 6)MGKTHIIGVGLDLGGTYTGTFITSHPSDEAEHRDHSSAFTVVNSEKLSFSSKSRTAVRHRVRSYKGFDLRRRLLLLVAEYQLLQKKQTLAPEERENLRIALSGYLKRRGYARTEAETDTSVLESLDPSVFSSAPSFTNFFNDSEPLNIQWEAIANSPETTKALNKELSGQKEADFKKYIKTSFPEYSAKEILANYVEGRRAILDASKYIANLQSLGHKHRSKYLSDILQDMKRDSRITRLSEAFGSTDNLWRIIGNISNLQERAVRWYFNDAKFEQGQEQLDAVKLKNVLVRALKYLRSDDKEWSASQKQIIQSLEQSGDVLDVLAGLDPDRTIPPYEDQNNRRPPEDQTLYLNPKALSSEYGEKWKSWANKFAGAYPLLTEDLTEILKNTDRKSRIKIRSDVLPDSDYRLAYILQRAFDRSIALDECSIRRTAEDFENGVVIKNEKLEDVLSGHQLEEFLEFANRYYQETAKAKNGLWFPENALLERADLHPPMKNKILNVIVGQALGVSPAEGTDFIEEIWNSKVKGRSTVRSICNAIENERKTYGPYFSEDYKFVKTALKEGKTEKELSKKFAAVIKVLKMVSEVVPFIGKELRLSDEAQSKFDNLYSLAQLYNLIETERNGFSKVSLAAHLENAWRMTMTDGSAQCCRLPADCVRPFDGFIRKAIDRNSWEVAKRIAEEVKKSVDFTNGTVKIPVAIEANSFNFTASLTDLKYIQLKEQKLKKKLEDIQRNEENQEKRWLSKEERIRADSHGICAYTGRPLDDVGEIDHIIPRSLTLKKSESIYNSEVNLIFVSAQGNQEKKNNIYLLSNLAKNYLAAVFGTSDLSQITNEIESTVLQLKAAGRLGYFDLLSEKERACARHALFLNSDSEARRAVIDVLGSRRKASVNGTQAWFVRSIFSKVRQALAAWTQETGNELIFDAISVPAADSSEMRKRFAEYRPEFRKPKVQPVASHSIDAMCIYLAACSDPFKTKRMGSQLAIYEPINFDNLFTGSCQVIQNTPRNFSDKTNIANSPIFKETIYAERFLDIIVSRGEIFIGYPSNMPFEEKPNRISIGGKDPFSILSVLGAYLDKAPSSEKEKLTIYRVVKNKAFELFSKVAGSKFTAEEDKAAKILEALHFVTVKQDVAATVSDLIKSKKELSKDSIENLAKQKGCLKKVEYSSKEFKFKGSLIIPAAVEWGKVLWNVFKENTAEELKDENALRKALEAAWPSSFGTRNLHSKAKRVFSLPVVATQSGAVRIRRKTAFGDFVYQSQDTNNLYSSFPVKNGKLDWSSPIIHPALQNRNLTAYGYRFVDHDRSISMSEFREVYNKDDLMRIELAQGTSSRRYLRVEMPGEKFLAWFGENSISLGSSFKFSVSEVFDNKIYTENAEFTKFLPKPREDNKHNGTIFFELVGPRVIFNYIVGGAASSLKEIFSEAGKERSPKKKRKVMcaCas9/NLS DNA sequence (SEQ ID NO: 7)ATGGAAAAGAAGCGGAAAGTCACCCTGGGCTTCGACCTGGGAATCGCCTCTGTTGGATGGGCCATCGTGGACAGCGAGACAAACCAGGTGTACAAGCTGGGCAGCAGACTGTTCGACGCCCCTGACACCAACCTGGAAAGAAGAACCCAGCGGGGCACCAGAAGGCTGCTGCGGAGAAGAAAGTACCGGAACCAGAAATTCTACAACCTGGTCAAGCGGACCGAGGTGTTCGGCCTGTCTAGCAGAGAGGCCATCGAGAACAGATTCAGAGAGCTGAGCATCAAGTACCCCAACATCATCGAGCTGAAAACAAAGGCCCTGAGCCAAGAAGTGTGCCCCGACGAGATTGCCTGGATTCTGCACGACTACCTGAAGAACCGGGGCTACTTCTACGACGAGAAAGAGACAAAAGAGGACTTCGACCAGCAGACCGTGGAATCCATGCCTAGCTACAAGCTGAACGAGTTCTACAAGAAGTACGGCTACTTCAAAGGCGCCCTGTCTCAGCCTACCGAGAGCGAGATGAAGGACAACAAGGACCTGAAAGAGGCATTCTTCTTCGACTTCTCCAACAAAGAGTGGCTGAAAGAGATCAACTACTTCTTCAACGTGCAGAAGAACATCCTGAGCGAGACATTCATCGAAGAGTTCAAGAAGATTTTCAGCTTCACCCGGGACATCAGCAAAGGCCCAGGCAGCGACAATATGCCCTCTCCTTACGGCATCTTCGGCGAGTTCGGCGACAATGGCCAAGGCGGCAGATACGAGCACATCTGGGACAAGAACATCGGCAAGTGCAGCATCTTCACCAACGAGCAGAGAGCCCCTAAGTACCTGCCTAGCGCTCTGATCTTCAACTTCCTGAACGAGCTGGCCAACATCAGACTGTACAGCACCGACAAGAAGAATATCCAGCCTCTGTGGAAGCTGAGCAGCATCGATAAGCTGAATATCCTGCTGAACCTGTTCAACCTGCCTATCAGCGAGAAGAAGAAAAAGCTGACCAGCACCAACATCAACGACATCGTGAAGAAAGAGTCCATCAAGAGCATCATGCTGAGCGTCGAGGACATCGACATGATCAAGGATGAGTGGGCCGGCAAAGAACCCAACGTGTACGGCGTTGGACTGAGCGGCCTGAACATCGAGGAAAGCGCCAAAGAGAACAAGTTCAAGTTCCAAGACCTGAAGATCCTGAACGTCCTGATCAATCTGCTGGACAACGTGGGCATCAAGTTCGAGTTCAAGGACCGCAGCGACATCATCAAGAACCTGGAACTGCTGGATAACCTGTACCTGTTCCTGATCTACCAGAAAGAGAGCAACAACAAAGACAGCTCCATCGACCTGTTTATCGCCAAGAACAAGTCCCTGAATATCGAGAACCTGAAGCTCAAGCTCAAAGAGTTCCTGCTCGGAGCCGGCAACGAGTTCGAGAACCACAACAGCAAGACCCACAGCCTGTCCAAGAAGGCCATTGACGCCATCCTGCCTAAGCTGCTCGACAACAACGAAGGCTGGAATCTGGAAGCCATCAAGAATTACGACGAGGAAATCAAGAGCCAGATCGAGGACAACTCCAGCCTGATGGCCAAGCAGGATAAGAAGTACCTGAACGACAACTTCCTCAAGGATGCCATTCTGCCGCCAAACGTGAAAGTGACCTTCCAGCAGGCCATCCTCATCTTCAACAAGATCATCCAGAAGTTCAGCAAGGATTTCGAGATCGACAAGGTCGTGATCGAACTGGCCAGAGAGATGACCCAGGACCAAGAGAACGACGCCCTGAAGGGAATCGCTAAGGCCCAGAAGTCCAAGAAAAGCCTGGTGGAAGAGAGACTCGAAGCCAACAACATCGACAAGAGCGTGTTCAACGATAAGTACGAGAAGCTTATCTACAAGATTTTCCTGTGGATCAGCCAGGACTTTAAGGACCCCTACACCGGCGCCAAGATCAGCGCCAATGAGATCGTGGATAACAAGGTGGAAATCGACCACATCATCCCTTACAGCCTGTGCTTCGACGACAGCAGCGCCAACAAAGTGCTGGTGCACAAGCAGAGCAATCAAGAGAAGTCTAACAGCCTGCCGTACGAGTACATCAAGCAGGGCCACTCCGGCTGGAACTGGGACGAGTTCACCAAATACGTGAAGCGGGTGTTCGTGAACAACGTGGACTCTATCCTGAGCAAGAAAGAGCGCCTGAAGAAGTCCGAGAATCTGCTGACCACCAGCTACGACGGCTATGAGAAGCTGGGCTTCCTGGCCAGAAACCTGAATGACACCAGATACGCCACCATCCTGTTCCGGGACCAGCTGAACAATTACGCCGAGCACCACCTGATCGATAACAAGAAAATGTTCAAAGTGATCGCCATGAACGGGGCCGTGACCAGCTTCATCCGGAAGAACATGAGCTACGACAACAAGCTGCGGCTGAAGGACAGAAGCGACTTCAGCCACCACGCCTACGACGCCGCCATCATTGCCCTGTTCAGCAACAAGACCAAGACGCTGTACAACCTGATTGACCCCAGCCTGAACGGCATCATCAGCAAGAGAAGCGAAGGCTATTGGGTCATCGAGGATCGGTACACAGGCGAGATCAAAGAGCTTAAGAAAGAGGATTGGACCTCTATCAAGAACAATGTGCAGGCCCGGAAGATCGCCAAAGAAATCGAGGAATATCTGATCGACCTGGACGATGAGGTGTTCTTCAGCCGGAAAACTAAGCGCAAGACCAACCGGCAGCTGTACAATGAGACAATCTACGGAATCGCCGCCAAGACCGACGAGGACGGCATCACCAACTACTACAAGAAAGAAAAGTTCTCCATCCTGGACGACAAGGACATCTACCTGCGGCTGCTGAGAGAACGCGAGAAGTTCGTGATCAACCAGAGCAACCCCGAAGTGATCGACCAGATTATCGAGATCATCGAGAGCTACGGGAAAGAAAACAACATCCCCAGCCGCGACGAGGCCATCAATATCAAGTACACGAAGAACAAGATTAACTACAACCTCTACCTCAAGCAGTACATGCGGAGCCTGACCAAGAGCCTGGACCAGTTCAGCGAGGGCTTCATCAATCAGATGATCGCCAACAAGACGTTCGTGCTGTATAACCCCACCAAGAACACAACGCGGAAGATCAAGTTCCTGCGGCTCGTGAACGATGTGAAGATCAACGATATTCGCAAGAATCAAGTGATCAACAAGTTTAACGGGAAGAACAACGAGCCCAAGGCCTTCTACGAGAATATCAACAGCCTGGGCGCCATCGTGTTCAAGTCCTCCGCCAACAACTTCAAGACCCTGTCCATCAACACCCAGATCGCCATCTTCGGAGACAAGAACTGGGATATCGAGGATTTCAAGACCTACAACATGGAAAAAATCGAGAAGTACAAAGAGATATACGGCATCGACAAAACCTACAACTTCCACAGCTTTATCTTCCCCGGCACAATCCTGCTCGATAAGCAGAACAAAGAGTTCTACTACATCAGCAGCATCCAGACCGTGAACGACCAAATTGAGCTGAAGTTTCTGAACAAGATCGAGTTTAAGAACGACGACAACACCTCCGGGGCCAACAAGCCTCCTCGGAGACTGAGATTCGGCATTAAGTCCATCATGAACAACTACGAGCAGGTCGACATCAGCCCCTTCGGCATCAACAAGAAGATATTCGAGCCCAAGAAGAAGAGGAAG GTGMcaCas9/NLS protein sequence (SEQ ID NO: 8)MEKKRKVTLGFDLGIASVGWAIVDSETNQVYKLGSRLFDAPDTNLERRTQRGTRRLLRRRKYRNQKFYNLVKRTEVFGLSSREATENRFRELSIKYPNIIELKTKALSQEVCPDEIAWILHDYLKNRGYFYDEKETKEDFDQQTVESMPSYKLNEFYKKYGYFKGALSQPTESEMKDNKDLKEAFFFDFSNKEWLKEINYFFNVQKNILSETFIEEFKKIFSFTRDISKGPGSDNMPSPYGIFGEFGDNGQGGRYEHIWDKNIGKCSIFTNEQRAPKYLPSALIFNFLNELANIRLYSTDKKNIQPLWKLSSIDKLNILLNLFNLPISEKKKKLTSTNINDIVKKESIKSIMLSVEDIDMIKDEWAGKEPNVYGVGLSGLNIEESAKENKFKFQDLKILNVLINLLDNVGIKFEFKDRSDIIKNLELLDNLYLFLIYQKESNNKDSSIDLFIAKNKSLNIENLKLKLKEFLLGAGNEFENHNSKTHSLSKKAIDAILPKLLDNNEGWNLEAIKNYDEEIKSQIEDNSSLMAKQDKKYLNDNFLKDAILPPNVKVTFQQAILIFNKIIQKFSKDFEIDKVVIELAREMTQDQENDALKGIAKAQKSKKSLVEERLEANNIDKSVFNDKYEKLIYKIFLWISQDFKDPYTGAKISANEIVDNKVEIDHIIPYSLCFDDSSANKVLVHKQSNQEKSNSLPYEYIKQGHSGWNWDEFTKYVKRVFVNNVDSILSKKERLKKSENLLTTSYDGYEKLGFLARNLNDTRYATILFRDQLNNYAEHHLIDNKKMFKVIAMNGAVISFIRKNMSYDNKLRLKDRSDFSHHAYDAAIIALFSNKTKTLYNLIDPSLNGIISKRSEGYWVIEDRYTGEIKELKKEDWTSIKNNVQARKIAKEIEEYLIDLDDEVFFSRKTKRKTNRQLYNETIYGIAAKTDEDGITNYYKKEKFSILDDKDIYLRLLREREKFVINQSNPEVIDQIIETIESYGKENNIPSRDEAINIKYTKNKINYNLYLKQYMRSLTKSLDQFSEGFINQMIANKTFVLYNPTKNTTRKIKFLRLVNDVKINDIRKNQVINKFNGKNNEPKAFYENINSLGAIVFKSSANNFKILSINTQTAIFGDKNWDIEDFKTYNMEKIEKYKEIYGIDKTYNFHSFIFPGTILLDKQNKEFYYISSIQTVNDQIELKFLNKIEFKNDDNTSGANKPPRRLRFGIKSIMNNYEQVDISPFGINKKIFEPKKKRKVPKKKRKVMgaCas9/NLS DNA sequence (SEQ ID NO: 9)ATGAACAACAGCATCAAGAGCAAGCCCGAAGTGACCATCGGCCTGGATCTCGGCGTTGGCTCTGTTGGATGGGCCATCGTGGACAACGAGACAAACATCATCCACCACCTGGGCAGCAGACTGTTCAGCCAGGCCAAGACAGCTGAGGACAGGCGGTCTTTCAGAGGCGTGCGGAGACTGATCCGGCGGAGAAAGTACAAGCTGAAGAGATTCGTGAACCTGATCTGGAAGTACAACAGCTACTTCGGCTTCAAGAACAAAGAGGACATCCTGAACAACTACCAAGAGCAGCAGAAACTGCACAACACCGTGCTGAACCTGAAGCTCGAAGCCCTGAACGCCAAGATCGACCCCAAGGCTCTGAGCTGGATTCTGCACGACTACCTGAAGAACCGGGGCCACTTCTACGAGGACAACCGGGACTTCAACGTGTACCCCACAGAGGAACTGGCCAACTACTTCGACGAGTTCGGCTACTACAAGGGCATCATCGACAGCAAGAACGACGACGATGATAAGCTGGAAGAGGGCCTGACCAAGTACAAGTTCAGCAACCAGCACTGGCTGGAAGAAGTGAAGAAGGTCCTGAGCAACCAGACCGGCCTGCCTGAGAAGTTCAAAGAGGAATACGAGAGCCTGTTCAGCTACGTGCGGAACTACTCTGAAGGCCCTGGCAGCATCAACAGCGTGTCCCCATACGGCATCTATCACCTGGACGAGAAAGAGGGCAAAGTCGTCCAGAAGTATAACAACATCTGGGACAAGACCATCGGGAAGTGCAGCATCTTCCCCGACGAGTACAGAGCCCCTAAGAACAGCCCTATCGCCATGATCTTCAACGAGATCAACGAGCTGAGCACCATCCGGTCCTACAGCATCTACCTGACCGGCTGGTTCATCAATCAAGAGTTCAAGAAGGCCTACCTGAACAAGCTGCTGGACCTGCTGATCAAGACCAACAGCGAGAAGCCCATCGACGCCCGGCAGTTTAAGAAGCTGCGGGAAGAGACAATCGCCGAGAGCATCGGCAAAGAAACCCTGAAGGACGTGGAAAGCGAGGAAAAGCTGGAAAAGGACGACCACAAGTGGAAGCTGAAGGGCCTGAAGCTGAACACCAACGGCAAGATCCAGTACAACGACCTGTCTAGCCTGGCCAAGTTCGTGCACAAACTGAAGCAGCACCTGAAACTGGACTTTCTGCTGGAAGATCAGTACACCCCTCTGGACAAGATCAACTTCCTGCAGAGCCTGTACGTGTACCTGGGCAAGCACCTGAGATACAGCAACAGAGTGGACAGCGCCAACCTGAAAGAGTTCAGCGACAGCTCCCGGCTGTTCGAGAGAGTGCTGCAAGAGCAGAAGGACGGCCTGTTCAAGCTGTTTGAGCAGACCGACAAGGACGACGAGAAGATCCTGACACAGACCCACAGCCTGTCCACCAAGGCTATGCTGCTGGCCATCACCAGAATGACCAACCTGGACAATGACGAGGATAACCAGAAGAACAACGACAAAGGCTGGAACTTCGAGGCCATCAAGAACTTCGACCAGAAGTTCATCGACATCACCAAGACGAACAACAACCTGAGCCTGAAGCAGGACAAGCGCTACCTGGATGACCAGTTCATCAACGACGCCATTCTGAGCCCTGGCGTGAAGAGAATCCTGCGCGAGGCCACCAAGGTGTTCAACGCCATCCTCAAGCAGTTCTCCGAAGAGTACGACGTGACCAAGGTGGTCATCGAGCTGGCCAGAGAGCTGAGCGAAGAGAAAGAACTGGAAAACACCAAGAACTACAAGAAGCTTATCAAGAAGAACGGCGATAAGATCAGCGAGGGACTGAAAGCCCTGGGGATCGCCGAGGATAAGATCGAAGAGATCCTGAAGTCTCCCACCAAGTCCTACAAAGTGCTGCTGTGGCTGCAGCAGGACCACATCGATCCCTACAGCCAGAAAGAGATCGCCTTCGACGATATCCTGACCAAAACCGAAAAGACCGAGATCGACCACATCATTCCTTACTCCATCAGCTTCGACGACAGCAGCAGCAACAAACTGCTGGTGCTGGCCGAGTCCAATCAGGCCAAGTCCAACCAGACACCTTACGAGTTTATCAACTCCGGCAAGGCCGAGATCACCTGGGAAGTGTACGAGGCCTACTGCCACAAGTTCAAAAACGGCGACTCCAGCCTGCTGGACAGCACCCAGAGAAGCAAGAAATTCGCCAAGATGATGAAGACCGACACCAGCTCTAAGTACGACATCGGCTTTCTGGCCCGGAACCTGAACGACACCAGATACGCCACCATCGTGTTCCGGGACGCTCTGAAGGACTACGCCAACAACCACCTGGTGGAAGATAAGCCCATGTTCAAGGTCGTGTGCATCAACGGCGGCGTGACCAGCTTCCTGCGGAAGAACTTTGACCCCAAGTCTTGGTACGCCAAGAAGGACAGAGACAAGAACATTCACCACGCCGTGGACGCCAGCATCATCTCCATCTTCAGCAACGAGACTAAGACCCTGTTCAACCAGCTGACAAAGTTCGCCGACTACAAGCTGTTCAAGAATACCGACGGCTCTTGGAAGAAGATCGATCCTAAGACAGGCGTGGTGTCAGAAGTGACCGACGAGAATTGGAAGCAGATCCGCGTGCGCAACCAGGTGTCCGAGATCGCCAAAGTGATCGACAAGTACATCCAGGACAGCAACATCGAGCGGAAGGCCAGATACAGCCGGAAGATCGAGAACAAGACCAATATCAGCCTGTTTAACGACACCGTGTACTCCGCCAAGAAAGTGGGCTACGAGGATCAGATCAAGCGCAAGAACCTGAAAACCCTGGACATCCACGAGAGCGCCGAGGAAAACAAGAACAGCAAAGTGAAAAAGCAGTTCGTGTACCGGAAGCTCGTGAACGTGTCCCTGCTGAACAATGACAAGCTGGCCGACCTGTTCGCCGAGAAAGAAGATATTCTGATGTACCGGGCCAATCCGTGGGTCATCAACCTGGCCGAGCAGATTTTCAACGAGTACACCGAGAACAAAAAGATCAAGAGCCAGAACGTGTTCGAGAAGTACATGCTGGATCTGACCAAAGAGTTCCCCGAGAAGTTTAGCGAGGCCTTCGTGAAGTCCATGATCAGAAACAAGACCGCCATCATCTACAACGTCGAGAAGGATGTGGTGCACCGGATCAAGCGGCTGAAGATTCTGAGCAGCGAGCTGAAAGAAAACAAGTGGTCCAACGTGATCATCCGCTCCAAGAACGAGAGCGGCACCAAGCTGAGCTACCAGGACACCATCACTCTATCGCCCTGATGATCATGCGGAGCATCGACCCAACCGCCAAAAAACAGTACATCAGGGTGCCCCTGAACACCCTGAATCTGCACCTGGGCGACCAGGACTTCGACCTGCACAATATCGACGCCTATCTGAAGAAGCCTAAGTTCGTCAAGTACCTGAAGGCCAATGAGATCGGCGACGAGTATAAGCCTTGGCGCGTGCTGACAAGCGGCACACTGCTGATCCACAAGAAAGACAAGAAACTCATGTACATCAGCAGCTTCCAGAACCTCAACGACCTCATCGAGATCAAGAATCTGATCGAGACAGAGTACAAAGAAAACGTGGACTCAGACCCCAAGAAGAAGAAAAAGGCCAGCCAGATCCTGAGAAGCCTGAGCGTGATCCTGAACGATTACATCCTGCTGGATGCCAAGTATAACTTCGACATCCTGGGCCTGTCTAAGAACAAGATTGACGAGATCCTCAACAGCAAGCTGGACCTCGACAAGATTGCCAAGCCCAAGAAGAAGAGGAAGGTGMgaCas9/NLS protein sequence (SEQ ID NO: 10)MNNSIKSKPEVTIGLDLGVGSVGWAIVDNETNIIHHLGSRLFSQAKTAEDRRSFRGVRRLIRRRKYKLKRFVNLIWKYNSYFGFKNKEDILNNYQEQQKLHNTVLNLKLEALNAKIDPKALSWILHDYLKNRGHFYEDNRDFNVYPTEELANYFDEFGYYKGIIDSKNDDDDKLEEGLTKYKFSNQHWLEEVKKVLSNQTGLPEKFKEEYESLFSYVRNYSEGPGSINSVSPYGIYHLDEKEGKVVQKYNNIWDKTIGKCSIFPDEYRAPKNSPIAMIFNEINELSTIRSYSTYLTGWFINQEFKKAYLNKLLDLLIKTNSEKPIDARQFKKLREETIAESIGKETLKDVESEEKLEKDDHKWKLKGLKLNTNGKIQYNDLSSLAKFVHKLKQHLKLDFLLEDQYTPLDKINFLQSLYVYLGKHLRYSNRVDSANLKEFSDSSRLFERVLQEQKDGLFKLFEQTDKDDEKILTQTHSLSTKAMLLAITRMTNLDNDEDNQKNNDKGWNFEAIKNFDQKFIDITKTNNNLSLKQDKRYLDDQFINDAILSPGVKRILREATKVFNAILKQFSEEYDVTKVVIELARELSEEKELENTKNYKKLIKKNGDKISEGLKALGIAEDKIEEILKSPTKSYKVLLWLQQDHIDPYSQKEIAFDDILTKTEKTEIDHIIPYSISFDDSSSNKLLVLAESNQAKSNQTPYEFINSGKAEITWEVYEAYCHKFKNGDSSLLDSTQRSKKFAKMMKTDTSSKYDIGFLARNLNDTRYATIVFRDALKDYANNHLVEDKPMFKVVCINGGVTSFLRKNFDPKSWYAKKDRDKNIHHAVDASIISIFSNETKTLFNQLTKFADYKLFKNTDGSWKKIDPKTGVVSEVTDENWKQIRVRNQVSEIAKVIDKYIQDSNIERKARYSRKIENKTNISLFNDTVYSAKKVGYEDQIKRKNLKTLDIHESAEENKNSKVKKQFVYRKLVNVSLLNNDKLADLFAEKEDILMYRANPWVINLAEQIFNEYTENKKIKSQNVFEKYMLDLTKEFFEKFSEAFVKSMIRNKTAITYNVEKDVVHRIKRLKILSSELKENKWSNVIIRSKNESGTKLSYQDTINSIALMIMRSIDPTAKKQYIRVPLNTLNLHLGDQDFDLHNIDAYLKKPKFVKYLKANEIGDEYKPWRVLTSGTLLIHKKDKKLMYISSFQNLNDLIEIKNLIETEYKENVDSDPKKKKKASQILRSLSVILNDYILLDAKYNFDILGLSKNKIDEILNSKLDLDKIAKPKKKRKV Ag1Cas9/NLS DNA sequence (SEQ ID NO: 11)ATGCAGAACATCACCTTCAGCTTCGACGTGGGCTACGCCTCTATCGGATGGGCTGTTGTTCAGGCCCCTGCTCAGCCAGAGCAGGACCCTGGAATAGTGGCCTGTGGCACCGTGCTGTTCCCTAGCGACGATTGCCAGGCCTTCCAGCGGAGAAACTACCGGCGGCTGCGGAGGAACATCCGGTCCAGAAGAGTGCGGATCGAGCGGATCGGAAAGCTGCTGGTTCAGGCCGGAATCCTGACACCTGAGGAAAAGGCCACACCTGGACACCCCGCTCCATTCTTTTTGGCAGCCCAGGCTTGGCAGGGCATCAGACAACTGTCTCCACTGGAAGTGTGGCACATCCTGCGTTGGTACGCCCACAACAGAGGCTACGACAACAATGCCGCCTGGGCCACCGTGTCCACCAAAGAGGATACCGAGAAAGTCAACAACGCCCGGCACCTGATGCAGAAGTTTGGCGCCGAGACAATGTGCGCCACACTGTGCCATGCTATGGAACTGGACATGGACGTGCCCGATGCCGCCATGACAGTGTCTACACCAGCCTACAGAACCCTGAACAGCGCCTTTCCTAGAGATGTGGTGCAGAGAGAGGTGCTGGACATCCTGAGACACAGCGCCAGCCACATCAAAGAGCTGACCCCTGAGATCATCCGGCTGATCGCCCAGCAAGAGGATCTGAGCACAGAGCAGAGAAGCGAGCTGGCCGCCAAGGGAATTAGACTGGCCAGAAGATACCGGGGCAGCCTGCTGTTTGGACAGCTGCTGCCCAGATTCGACAACCGGATCATCAGACGGTGCCCCATCATCTGGGCCCACACATTTGAGCAGGCCAAGACCAGCGGCATGAGCGAGAAAGAAGCTCAGGCCCTGGCTGACAAGGTGGCCAAAGTGCCTACAGCCGACTGTCCCGAGTTCTACGCCTACAGATTCGCCCGCATCCTGAACAATCTGAGAGCCAACGGACTGCCCCTGCCTGTGGAAGTTCGCTGTGAACTGATGCAGGCCGCCAGAGCCGAGGGAAAACTGACAGCCGCCAAGATCAAGAAAGAAATCATGAGGCTGATGGGCGACGTCGAGAGCAACATCGACCACTACTTCCATCTGCACCCCGACAGCGAGGAAGCCCTGATTCTCGATCCCGCTATGGAATGCCTGCACCGGACCGGACTGTACGATGCCCTCAGCTCTGTCGTGCGAAGAGTGGCCCTGACCAGACTGCGGAGAGGCAAAATCTGTACCCCTGCCTACCTGCGGGACATGATGCTGAGACACGGCGAGGATACCCAGGCTCTGGATCTGGCCATTGCCAAGCAGCAGGGAAGAAAGGCCCCTCGGCCTAGAAAGAACGACACAGATGCCAGCGCCGACGCCAGCATTGCATGGCAAGATAAGCCCCTGGCTCCTAAGACAGCCTCTGGCAGAGCCCCTTATGCCAGACCAGTTCTGAGACAGGCCGTGGACGAGATCATGAATGGCGAGGACCCTACCAGGCCAGCTCTGGATGAACAGCATCCCGACGGCGAGGACAAGCCTTCTCACGGCTGTCTGTATGGCCTGCTGGACCCTGCCAGCAAAGAGAACGAGTACCTGAACAAGCTTCCCCTGGACGCCCTGACAAACAATCACCTCGTGCGGCACCGGATGCTGATCCTGGATAGACTGACCCAGGACCTCGTCAGAGAGTTCGCTGACGGCGATCCCAGCAGAGTGGAACGGTTCTGTATCGAAGTGGGCAGAGAGCTGAGCGCCTTCTCTGGCATGACCAGCAAGCAGATCCAGTCCGAGCTGAACGAGCGGATGAAGCACTTCAAGAGCGCCGTGGCCTATCTGGCCAAACACGCCCCTGATATGGCCACATCTGCCGGCCTGATCCGGAAGTGCAGAATCGCTATGGACATGAACTGGCAGTGCCCTTTCACCGGCCAGACCTACATGCCCTACGACCTGCCTAAGCTGGAACGCGAGCACATCGTGCCCTACGCCAACAGAAAGACAGATGCCCTGTCTGCCCTGGTGCTGACATGGCTGGCCGTGAACAAGATGAAGGGCAAGAGAACCGCCTACCAGTTTATCAAAGAGTGCGAGGGCCAGAGCGTGCCCGGCAGAAATCAGAATATCGTGTCCGTGAAGCAGTACGAGACATTCGTGGAAAAGCTGGACACCAAGGGCCACGCCGACGACGCCAAGAGAAAAAAGACCCGGAAGAAACTGATGATGGTGGACAGACTGAGCAGCCAGGGAACAAACGGCGAGTCTGAGCTGGATTTCACCGAGGGCATGATGACCCAGAGCAGCCACCTGATGAAGATCGCCGCTAGAGGCGTGCGGAAGAACTTTCCTCACGCCACCGTGGACATGATCCCTGGCGCTATTACTGGCACTGTGCGCAAGGCTTGGAAGGTGGCAGGATGCCTGGCCGGCATTTGTCCTGAAGCCGTCGATCCCGTGACACACAGAATCCAGGACAAAGAGACACTGCGGCGGCTGACCCATCTGCATCATGCACTGGATGCCTGCGTGCTGGGACTGATCCCTCACCTGATTCCAGAGCACAGATCCGGCCTGCTGAGAAAAGCTCTGGCCGCTAGAAGGCTGCCCGAGAATGTTCGGCAAGAGGTGGAAAGCGCCGTGTCCAAGCGGTACTACACCATCACAAAAGAGAGCAAACTGGAACTGCGGGATCTGCCCACCACACTGAAGAACTCTATCGCCGCCAAGCTGAGCGAGGGCAGAGTGGTGCAACACATCCCTGCCGATATGAGCGGAGCCAAGCTGGAAGAGACAATCTGGGGAATTGCCCCTGGCCAGCACATCGACGACAATAGCGAGGTGGTCATCCGGCAGAAGTCCCTGAGCATCGGCAAGGACGGCAACAGAATCAGAACCAGAAAGACCGACAAGCAGGGCAACCCCATCACCGAGAAGGCCTCTAAGCTCGTGGGCATCAAGCCTACCGGCACCAGCAAACTGCAGCCCATCAGAGGCGTGATCATCATCAAGGACAACTTCGCCATTGCTCTGGACCCCGTGCCAACCATGATTCCCCACCACAACGTGTACAAGCGGCTGGAAGAACTGCGGAAGCTGAACCACGGTAGACATGTGCGGCTGCTGAAAAAGGGCATGCTGATCAGGCTGAGCCACCAGAAGTCCGGCGACAAGAACGGCATGTGGAAAGTGCGGAGCATCCAGGACCAGGGCTCCTCTGGCCTGAAAGTGAATCTGCAGAGGCCCTACTACGCCGGCAAGATCGAGGACACCAGAACCGAGAATTGGAAGAACGTGTCCATCAAGGCCCTGCTGAGCCAAGGCATGGAAATCCTGCCAACCACCTACTGCGGCACCACACCTCCCAAGAAGAAGAGGAAGGTGAg1Cas9/NLS protein sequence (SEQ ID NO: 12)MQNITFSFDVGYASIGWAVVQAPAQPEQDPGIVACGTVLFPSDDCQAFQRRNYRRLRRNIRSRRVRIERIGKLLVQAGILTPEEKATPGHPAPFFLAAQAWQGIRQLSPLEVWHILRWYAHNRGYDNNAAWATVSTKEDTEKVNNARHLMQKFGAETMCATLCHAMELDMDVPDAAMTVSTPAYRTLNSAFPRDVVQREVLDILRHSASHIKELTPEIIRLIAQQEDLSTEQRSELAAKGIRLARRYRGSLLFGQLLPRFDNRIIRRCPIIWAHTFEQAKTSGMSEKEAQALADKVAKVPTADCPEFYAYRFARILNNLRANGLPLPVEVRCELMQAARAEGKLTAAKIKKEIMRLMGDVESNIDHYFHLHPDSEEALILDPAMECLHRTGLYDALSSVVRRVALTRLRRGKICTPAYLRDMMLRHGEDTQALDLAIAKQQGRKAPRPRKNDTDASADASIAWQDKPLAPKTASGRAPYARPVLRQAVDEIMNGEDPIRPALDEQHPDGEDKPSHGCLYGLLDPASKENEYLNKLPLDALTNNHLVRHRMLILDRLTQDLVREFADGDPSRVERFCIEVGRELSAFSGMTSKQIQSELNERMKHFKSAVAYLAKHAPDMATSAGLIRKCRIAMDMNWQCPFTGQTYMPYDLPKLEREHIVPYANRKTDALSALVLTWLAVNKMKGKRTAYQFIKECEGQSVPGRNQNIVSVKQYETFVEKLDTKGHADDAKRKKTRKKLMMVDRLSSQGTNGESELDFTEGMMTQSSHLMKIAARGVRKNFPHATVDMIPGAITGTVRKAWKVAGCLAGICPEAVDPVTHRIQDKETLRRLTHLHHALDACVLGLIPHLIPEHRSGLLRKALAARRLPENVRQEVESAVSKRYYTITKESKLELRDLPTTLKNSIAAKLSEGRVVQHIPADMSGAKLEETIWGIAPGQHIDDNSEVVIRQKSLSIGKDGNRIRTRKTDKQGNPITEKASKLVGIKPIGTSKLQPIRGVIIIKDNFAIALDPVPTMIPHHNVYKRLEELRKLNHGRHVRLLKKGMLIRLSHQKSGDKNGMWKVRSIQDQGSSGLKVNLQRPYYAGKIEDTRTENWKNVSIKALLSQGMEILPTTYCGTTPPKKKRKVAmuCas9/NLS DNA sequence (SEQ ID NO: 13)ATGAGCAGAAGCCTGACCTTCAGCTTCGACATCGGCTACGCCTCTATCGGCTGGGCCGTGATTGCCTCTGCCAGCCACGATGATGCCGATCCTAGCGTGTGTGGCTGTGGCACCGTGCTGTTCCCCAAGGATGATTGCCAGGCCTTCAAGCGGAGAGAGTACCGGCGGCTGCGGAGAAACATCCGGTCCAGAAGAGTGCGGATCGAGCGGATTGGTAGACTGCTGGTGCAGGCCCAGATCATCACCCCTGAGATGAAGGAAACCAGCGGACACCCCGCTCCATTCTACCTGGCATCTGAGGCCCTGAAGGGCCACAGAACACTGGCCCCTATTGAACTGTGGCATGTGCTGCGTTGGTACGCCCACAACAGAGGCTACGACAACAACGCCAGCTGGTCCAACAGCCTGTCTGAGGATGGTGGCAACGGCGAGGATACCGAGAGAGTGAAACACGCCCAGGACCTGATGGACAAGCACGGCACAGCTACAATGGCCGAGACAATCTGCAGAGAGCTGAAGCTGGAAGAGGGCAAAGCCGACGCTCCTATGGAAGTGTCTACCCCTGCCTACAAGAACCTGAACACCGCCTTTCCACGGCTGATCGTGGAAAAAGAAGTGCGGAGAATCCTGGAACTGAGCGCCCCTCTGATCCCTGGACTGACAGCCGAGATCATCGAGCTGATCGCCCAGCATCACCCTCTGACCACTGAACAGAGAGGCGTGCTGCTCCAGCACGGCATTAAGCTGGCCAGAAGATACAGAGGCAGCCTGCTGTTCGGCCAGCTGATCCCTAGATTCGACAACAGGATCATCAGCAGATGCCCCGTGACATGGGCCCAAGTGTATGAGGCCGAGCTGAAGAAGGGCAACAGCGAGCAGTCTGCCAGAGAGAGAGCCGAGAAGCTGAGCAAGGTGCCCACCGCCAATTGTCCCGAGTTCTACGAGTACCGGATGGCCAGAATCCTGTGCAACATCAGAGCCGACGGCGAGCCTCTGAGCGCCGAGATTAGACGCGAGCTGATGAACCAGGCCAGACAAGAGGGAAAGCTGACCAAGGCCAGCCTGGAAAAGGCCATCTCTAGCCGGCTGGGCAAAGAAACCGAGACAAACGTGTCCAACTACTTCACACTGCACCCCGACAGCGAGGAAGCCCTGTATCTGAATCCTGCCGTGGAAGTGCTGCAGAGAAGCGGCATCGGCCAGATTCTGAGCCCCAGCGTGTACAGAATCGCCGCCAACAGACTGCGGAGAGGCAAGAGCGTGACCCCTAACTACCTGCTGAATCTGCTGAAGTCCAGAGGCGAGTCTGGCGAGGCCCTGGAAAAAAAGATCGAGAAAGAGTCCAAGAAGAAAGAGGCCGACTACGCCGACACACCCCTGAAGCCTAAGTACGCCACAGGCAGAGCCCCTTACGCCAGAACCGTGCTGAAGAAAGTGGTGGAAGAGATCCTGGATGGCGAGGACCCTACCAGACCTGCTAGAGGCGAAGCTCACCCTGACGGCGAACTGAAAGCCCACGATGGCTGCCTGTACTGCCTGCTGGATACCGACAGCAGCGTGAACCAGCACCAGAAAGAGCGGAGACTGGACACCATGACCAACAACCACCTCGTGCGGCACCGGATGCTGATCCTGGACAGACTCCTGAAGGATCTGATCCAGGACTTCGCCGACGGCCAGAAGGACAGAATCAGCAGAGTGTGCGTGGAAGTCGGCAAAGAGCTGACCACCTTCAGCGCTATGGACAGCAAGAAGATCCAGCGGGAACTGACCCTGCGGCAGAAGTCTCATACCGACGCCGTGAACAGACTGAAGAGAAAGCTTCCAGGCAAGGCCCTGAGCGCCAACCTGATCAGAAAGTGCAGAATCGCAATGGACATGAACTGGACATGCCCCTTCACCGGCGCCACATATGGCGATCACGAGCTGGAAAATCTGGAACTGGAACACATCGTGCCCCACAGCTTCAGACAGAGCAATGCCCTGTCTAGCCTGGTGCTGACATGGCCTGGCGTGAACAGGATGAAGGGACAGAGAACCGGCTACGACTTCGTGGAACAAGAGCAAGAGAACCCCGTGCCTGACAAGCCCAACCTGCACATCTGCAGCCTGAACAACTATCGCGAGCTGGTGGAAAAGCTGGACGACAAGAAGGGACACGAGGACGACAGACGGCGGAAGAAGAAAAGAAAGGCCCTGCTGATGGTCCGAGGCCTGTCTCACAAACACCAGAGCCAGAACCACGAGGCCATGAAAGAAATCGGCATGACCGAGGGCATGATGACCCAGAGCAGCCACCTGATGAAGCTGGCCTGCAAGAGCATCAAGACCAGCCTGCCTGACGCTCACATCGACATGATTCCAGGCGCCGTGACTGCCGAAGTTCGCAAAGCCTGGGATGTGTTCGGCGTGTTCAAAGAACTGTGCCCCGAAGCCGCCGATCCTGACTCTGGCAAGATCCTGAAAGAGAACCTGCGGAGCCTGACTCATCTGCATCACGCCCTGGATGCCTGTGTGCTGGGACTGATCCCCTACATCATCCCCGCTCACCACAATGGCCTGCTGAGAAGAGTCCTGGCCATGCGCAGAATCCCCGAGAAACTGATCCCTCAAGTGCGGCCCGTGGCCAACCAGAGACACTACGTGCTGAACGACGACGGCCGGATGATGCTGAGGGATCTGAGTGCCAGCCTGAAAGAAAACATCCGCGAGCAGCTGATGGAACAGCGAGTGATCCAGCACGTGCCCGCTGATATGGGCGGAGCACTGCTCAAAGAAACAATGCAGCGGGTGCTGAGCGTGGACGGCTCTGGCGAAGATGCTATGGTGTCCCTGTCTAAGAAGAAGGACGGCAAGAAAGAGAAGAATCAAGTCAAGGCCTCCAAGCTCGTGGGAGTGTTTCCTGAGGGCCCCAGCAAGCTGAAAGCTCTGAAGGCCGCCATCGAGATCGACGGCAATTATGGCGTGGCACTGGACCCCAAGCCTGTGGTCATCAGACACATCAAGGTGTTCAAGAGGATCATGGCCCTCAAAGAGCAGAACGGCGGCAAGCCAGTGCGCATCCTGAAAAAGGGCATGCTGATTCACCTGACCAGCAGCAAGGACCCTAAGCACGCTGGCGTTTGGAGAATCGAGAGCATCCAGGACAGCAAAGGCGGCGTGAAACTGGACCTGCAGAGGGCTCATTGCGCCGTGCCTAAGAACAAGACCCACGAGTGCAATTGGAGAGAGGTGGACCTGATCTCCCTGCTGAAAAAGTACCAGATGAAGCGCTACCCCACCAGCTACACCGGCACACCTAGACCCAAGAAGAAGAGGAAGGTGAmuCas9/NLS protein sequence (SEQ ID NO: 14)MSRSLTFSFDIGYASIGWAVIASASHDDADPSVCGCGTVLFPKDDCQAFKRREYRRLRRNIRSRRVRIERIGRLLVQAQIITPEMKETSGHPAPFYLASEALKGHRTLAPIELWHVLRWYAHNRGYDNNASWSNSLSEDGGNGEDTERVKHAQDLMDKHGTATMAETICRELKLEEGKADAPMEVSTPAYKNLNTAFPRLIVEKEVRRILELSAPLIPGLTAEIIELIAQHHPLTTEQRGVLLQHGIKLARRYRGSLLFGQLIPRFDNRIISRCPVTWAQVYEAELKKGNSEQSARERAEKLSKVPTANCPEFYEYRMARILCNIRADGEPLSAEIRRELMNQARQEGKLTKASLEKAISSRLGKETETNVSNYFTLHPDSEEALYLNPAVEVLQRSGIGQILSPSVYRIAANRLRRGKSVTPNYLLNLLKSRGESGEALEKKIEKESKKKEADYADTPLKPKYATGRAPYARTVLKKVVEEILDGEDPTRPARGEAHPDGELKAHDGCLYCLLDTDSSVNQHQKERRLDTMTNNHLVRHRMLILDRLLKDLIQDFADGQKDRISRVCVEVGKELTTFSAMDSKKIQRELTLRQKSHTDAVNRLKRKLPGKALSANLIRKCRIAMDMNWTCPFTGATYGDHELENLELEHIVPHSFRQSNALSSLVLTWPGVNRMKGQRTGYDFVEQEQENPVPDKPNLHICSLNNYRELVEKLDDKKGHEDDRRRKKKRKALLMVRGLSHKHQSQNHEAMKEIGMTEGMMTQSSHLMKLACKSIKTSLPDAHIDMIPGAVTAEVRKAWDVFGVFKELCPEAADPDSGKILKENLRSLTHLHHALDACVLGLIPYIIPAHHNGLLRRVLAMRRIPEKLIPQVRPVANQRHYVLNDDGRMMLRDLSASLKENIREQLMEQRVIQHVPADMGGALLKETMQRVLSVDGSGEDAMVSLSKKKDGKKEKNQVKASKLVGVFPEGPSKLKALKAAIEIDGNYGVALDPKPVVIRHIKVFKRIMALKEQNGGKPVRILKKGMLIHLTSSKDPKHAGVWRIESIQDSKGGVKLDLQRAHCAVPKNKTHECNWREVDLISLLKKYQMKRYPTSYTGTPRPKKKRKV SEQ ID NO: 15. OkiCas9/NLS DNAATGGCCAGAGATTACAGCGTCGGCCTGGATATCGGCACCTCTTCTGTTGGATGGGCCGCCATCGACAACAAGTACCACCTGATCCGGGCCAAGAGCAAGAACCTGATTGGCGTGCGGCTGTTCGATAGCGCCGTGACCGCCGAGAAGAGAAGAGGCTACAGAACCACCAGACGGCGGCTGAGCAGACGGCATTGGAGACTGAGACTGCTGAACGACATCTTCGCCGGACCTCTGACCGATTTCGGCGACGAGAATTTCCTGGCCAGACTGAAGTACAGCTGGGTTCACCCTCAAGACCAGAGCAATCAGGCCCACTTTGCCGCCGGACTGCTGTTCGACAGCAAAGAGCAGGACAAGGACTTCTACCGGAAGTACCCCACCATCTATCACCTGAGACTGGCCCTGATGAACGACGACCAGAAGCACGACCTGAGAGAGGTGTACCTGGCCATCCACCACCTGGTCAAGTACAGAGGCCACTTCCTGATCGAGGGCGACGTGAAAGCCGACAGCGCCTTTGATGTGCACACCTTCGCCGACGCCATCCAGAGATACGCCGAGAGCAACAACTCCGACGAGAACCTGCTGGGCAAGATCGACGAGAAGAAGCTGAGCGCTGCCCTGACCGATAAGCACGGCAGCAAAAGCCAGAGAGCCGAGACAGCCGAAACCGCCTTCGACATCCTGGACCTGCAGTCCAAGAAGCAGATCCAGGCCATCCTGAAGTCCGTCGTGGGCAACCAGGCCAATCTGATGGCCATTTTTGGCCTGGACAGCAGCGCCATCAGCAAGGACGAGCAGAAGAACTACAAGTTCAGCTTCGACGACGCCGACATCGATGAGAAGATCGCCGATTCTGAGGCCCTGCTGAGCGATACCGAGTTCGAGTTCCTGTGCGATCTGAAGGCCGCCTTTGACGGCCTGACACTGAAAATGCTGCTGGGCGACGACAAGACCGTGTCCGCTGCTATGGTTCGACGGTTCAACGAGCACCAGAAGGACTGGGAGTACATCAAGAGCCACATCCGGAACGCCAAGAACGCCGGCAATGGCCTGTACGAGAAGTCTAAGAAGTTCGACGGCATCAACGCCGCCTATCTGGCTCTGCAGTCCGACAACGAGGACGACAGAAAGAAGGCCAAGAAGATTTTCCAGGACGAGATCAGCTCCGCCGACATTCCCGATGATGTGAAGGCCGATTTCCTGAAGAAGATTGACGACGATCAGTTCCTGCCTATCCAGCGGACCAAGAACAACGGCACAATCCCTCACCAGCTGCACCGGAACGAGCTGGAACAGATCATCGAGAAGCAGGGGATCTACTACCCATTCCTGAAGGACACCTACCAAGAGAACAGCCACGAGCTGAACAAAATCACAGCCCTGATCAACTTCAGGGTGCCCTACTACGTGGGCCCTCTGGTGGAAGAGGAACAGAAAATCGCCGACGACGGCAAGAACATCCCCGATCCTACCAACCACTGGATGGTCCGAAAGTCCAACGACACCATCACACCCTGGAACCTGAGCCAGGTGGTCGACCTGGATAAGAGCGGCAGAAGATTCATCGAGCGGCTGACCGGCACCGATACCTATCTGATCGGAGAGCCCACACTGCCCAAGAACAGCCTGCTGTACCAGAAATTCGACGTGCTGCAAGAACTGAACAACATCCGCGTGTCCGGCAGACGGCTGGACATTAGAGCCAAGCAGGATGCCTTCGAGCACCTGTTCAAGGTGCAGAAAACCGTGTCTGCTACCAATCTGAAGGACTTCCTGGTGCAAGCCGGCTACATCAGCGAGGACACCCAGATTGAAGGACTCGCCGACGTGAACGGAAAGAACTTCAACAACGCCCTGACCACCTACAACTACCTGGTGTCTGTGCTGGGCCGCGAGTTCGTGGAAAACCCCAGCAACGAGGAACTGCTGGAAGAGATTACCGAGCTGCAGACCGTGTTCGAGGACAAGAAGGTGCTGCGGAGACAGCTGGATCAGCTGGACGGACTGAGCGACCACAACAGAGAGAAGCTTTCCCGGAAGCACTACACCGGCTGGGGCAGAATCAGCAAGAAGCTGCTGACCACCAAGATCGTGCAGAACGCCGACAAGATCGATAACCAGACCTTCGATGTGCCCCGGATGAACCAGAGCATCATCGACACCCTGTACAACACCAAGATGAACCTGATGGAAATCATCAACAATGCCGAGGATGACTTCGGCGTCAGAGCCTGGATCGACAAGCAGAACACCACCGATGGCGACGAGCAGGACGTGTACAGCCTGATCGATGAACTGGCTGGCCCCAAAGAGATCAAGCGGGGCATCGTGCAGTCCTTTAGAATCCTGGACGACATCACCAAGGCCGTGGGCTACGCCCCTAAACGGGTGTACCTCGAATTTGCCAGAAAGACCCAAGAGAGCCACCTGACCAACAGCCGGAAGAACCAGCTGAGCACCCTGCTGAAGAATGCCGGCCTGTCTGAGCTGGTCACACAGGTGTCCCAGTATGATGCCGCCGCTCTGCAGAACGACCGGCTGTATCTTTACTTCCTGCAGCAAGGCAAGGACATGTACTCCGGCGAGAAGCTGAATCTGGACAACCTGAGCAACTACGACATCGACCACATCATCCCTCAGGCTTACACCAAGGACAACAGCCTGGACAACAGAGTGCTGGTGTCCAATATCACCAACCGGCGGAAGTCCGACAGCAGCAACTATCTGCCCGCTCTGATCGATAAGATGCGGCCCTTTTGGAGCGTGCTGAGCAAGCAGGGGCTGCTGTCTAAGCACAAGTTCGCCAACCTGACCAGAACCAGAGACTTCGACGATATGGAAAAAGAGCGGTTTATCGCCCGCAGCCTGGTGGAAACCCGGCAGATCATTAAGAACGTGGCCAGCCTGATTGACAGCCACTTCGGCGGAGAGACAAAAGCCGTGGCCATTAGAAGCAGCCTGACAGCCGACATGCGGAGATACGTGGACATCCCCAAGAACCGGGACATCAACGACTACCACCACGCCTTCGATGCCCTGCTGTTTAGCACAGTGGGCCAGTACACCGAGAACAGCGGCCTGATGAAGAAGGGCCAGCTGTCCGATTCTGCCGGCAACCAGTACAATCGGTACATCAAAGAGTGGATTCACGCCGCCAGGCTGAACGCACAGTCCCAGAGAGTGAACCCCTTCGGCTTTGTCGTGGGCTCCATGAGAAATGCTGCCCCTGGCAAGCTGAACCCCGAGACAGGGGAGATCACCCCAGAGGAAAACGCCGACTGGTCTATCGCCGACCTGGACTACCTGCACAAAGTGATGAATTTCCGGAAGATCACCGTGACCAGGCGGCTGAAGGATCAGAAAGGACAGCTGTACGACGAGAGCAGATACCCCTCCGTGCTGCACGACGCCAAGTCTAAGGCCAGCATCAACTTTGACAAGCACAAGCCCGTGGACCTGTACGGCGGCTTTAGCTCTGCCAAGCCTGCCTATGCCGCACTGATCAAGTTCAAGAACAAGTTCCGGCTGGTCAACGTGCTGCGGCAGTGGACCTACAGCGACAAGAACTCCGAGGACTATATCCTTGAGCAGATCAGAGGCAAGTACCCTAAGGCCGAGATGGTGCTGTCTCACATCCCTTACGGCCAGCTGGTCAAGAAAGATGGCGCCCTGGTCACCATCTCTAGCGCCACAGAGCTGCACAACTTTGAGCAGCTGTGGCTGCCTCTGGCCGACTACAAGCTGATCAACACACTGCTTAAGACCAAAGAGGACAACCTCGTCGATATCCTGCACAACCGGCTGGATCTCCCCGAGATGACAATCGAGAGCGCCTTCTACAAAGCCTTCGACTCCATCCTGAGCTTCGCCTTCAACAGATACGCCCTGCACCAGAACGCCCTCGTGAAACTGCAGGCCCACAGGGACGATTTCAATGCCCTGAACTACGAGGATAAGCAGCAGACCCTGGAAAGGATTCTGGACGCTCTGCATGCCTCTCCAGCCAGCAGCGACCTGAAGAAAATCAACCTGTCCAGCGGCTTCGGCCGGCTGTTTTCCCCTAGCCACTTTACCCTGGCCGACACCGACGAGTTCATCTTCCAGAGCGTGACCGGCCTGTTCAGCACCCAGAAAACAGTGGCTCAGCTGTATCAAGAGACAAAGCCCAAGAAGAAGAGGAAGGTGOkiCas9/NLS protein sequence (SEQ ID NO: 16)MARDYSVGLDIGTSSVGWAAIDNKYHLIRAKSKNLIGVRLFDSAVTAEKRRGYRTTRRRLSRRHWRLRLLNDIFAGPLTDFGDENFLARLKYSWVHPQDQSNQAHFAAGLLFDSKEQDKDFYRKYPTIYHLRLALMNDDQKHDLREVYLAIHHLVKYRGHFLIEGDVKADSAFDVHTFADAIQRYAESNNSDENLLGKIDEKKLSAALTDKHGSKSQRAETAETAFDILDLQSKKQIQAILKSVVGNQANLMAIFGLDSSAISKDEQKNYKFSFDDADIDEKIADSEALLSDTEFEFLCDLKAAFDGLTLKMLLGDDKTVSAAMVRRFNEHQKDWEYIKSHIRNAKNAGNGLYEKSKKFDGINAAYLALQSDNEDDRKKAKKIFQDEISSADIPDDVKADFLKKIDDDQFLPIQRTKNNGTIPHQLHRNELEQIIEKQGIYYPFLKDTYQENSHELNKITALINFRVPYYVGPLVEEEQKIADDGKNIPDPTNHWMVRKSNDTITPWNLSQVVDLDKSGRRFIERLTGTDTYLIGEPTLPKNSLLYQKFDVLQELNNIRVSGRRLDIRAKQDAFEHLFKVQKTVSATNLKDFLVQAGYISEDTQIEGLADVNGKNFNNALTTYNYLVSVLGREFVENPSNEELLEEITELQTVFEDKKVLRRQLDQLDGLSDHNREKLSRKHYTGWGRISKKLLTTKIVQNADKIDNQTFDVPRMNQSIIDTLYNTKMNLMEIINNAEDDFGVRAWIDKQNTTDGDEQDVYSLIDELAGPKEIKRGIVQSFRILDDITKAVGYAPKRVYLEFARKTQESHLTNSRKNQLSTLLKNAGLSELVTQVSQYDAAALQNDRLYLYFLQQGKDMYSGEKLNLDNLSNYDIDHIIPQAYTKDNSLDNRVLVSNITNRRKSDSSNYLPALIDKMRPFWSVLSKQGLLSKHKFANLTRTRDFDDMEKERFIARSLVETRQIIKNVASLIDSHFGGETKAVAIRSSLTADMRRYVDIPKNRDINDYHHAFDALLFSTVGQYTENSGLMKKGQLSDSAGNQYNRYIKEWIHAARLNAQSQRVNPFGFVVGSMRNAAPGKLNPETGEITPEENADWSIADLDYLHKVMNFRKITVTRRLKDQKGQLYDESRYPSVLHDAKSKASINFDKHKPVDLYGGFSSAKPAYAALIKFKNKFRLVNVLRQWTYSDKNSEDYILEQIRGKYPKAEMVLSHIPYGQLVKKDGALVTISSATELHNFEQLWLPLADYKLINTLLKTKEDNLVDILHNRLDLPEMTIESAFYKAFDSILSFAFNRYALHQNALVKLQAHRDDFNALNYEDKQQTLERILDALHASPASSDLKKINLSSGFGRLFSPSHFTLADTDEFIFQSVTGLFSTQKTVAQLYQETKPKKKRKV BboCas9/NLS DNA sequence (SEQ ID NO: 17)ATGAGCCAGCACCGGCGGTATAGAATCGGCATCGACGTGGGCCTGAATAGCGTTGGACTGGCCGCCGTGGAAATCGACGCCAACCACGACAATCCTCTGGACGAGATCCCCATCAGCATCCTGAATGCCCAGAGCGTGATCCACGATGGCGGAGTGGACCCTGATGAGGCCAAGTCTGCTACAAGCAGACGGGCTTCTGCTGGCGTGGCCAGAAGAACAAGACGGCTGCACAAGAGCAAGCGGCAGAGACTGGCCAAGCTGGACGAGGTGCTGAATGAGCTGGGCTACCCCGTGGAAGATGAGAGCCAGTTTCCAGCCGGCAGCAACCCCTATATCGCTTGGCAAGTGCGGGCCAAACTGGCCGAGACATTCATCCCCGACGTGGAAACCCGGAAGCGGATGATCTCTATCGCCATCCGGCACATTGCCCGGCATAGAGGATGGCGGAATCCCTACTCTTCTGTGGCCGACGCCGAGCGGATGAGCCATACACCTTCTCCATTCATGGTGGAATACGCCAAGAAGCTGGACTTCGAGATCAACGACAGACGGACCAACGGCTTCTATCACAGCCCTTGGCAGAGCGTGGACGAGGAAGGCAAGAGACTGAGCAAGAGCGAGCTGGAAAAGCAGCCCAAGATCGAGGACTGGAACGACAACCCCATCAACGGCAAGACAATCGCCCAGCTGGTCGTGTCCTCTCTGGAACCCCAGACCAAGATCAGACGGGATCTGACACACGGCCTGCAGACCGAGAGCACCCTGAATATCCAGACAGAGAAGCTGCACCAGAGCGACTACATCCACGAACTGGAAACCATCTTCGAGCGGCAGCACGTGGACCAGACAACCCAAGAACAGCTGCTGGAAGCCACCTTCCACACCAAGAATCCTAAGGCCGTGGGAGCCGCCGCTAAGCTCGTTGGAAAAGATGCCCTGGACAGCCGGTACTACAGAGCCAGCAGAGCCACACCAGCCTTCGAAGAGTACAGAGTGATGGCCGCCATCGACACCCTGCGGATTAGAGAGCACGGCACCGAGAGACAGCTGACCACCGACGAGAGAAGAAAGCTGTTCGACTTCATCAAGGGGCTGCCCAGCAAAAAGACCAAGAACGAGCCCAGCATCAGCTCCCTGACCTGGGGAGATGTGGCCGATTTTCTGGGCATCCAGCGGATCGATCTGAGAGGCCTGGGCTCTCTGAAAGACGGCGAACCTGTGTCTGCCAAGCAGCCTCCTGTGATCGAGACAAACGACATCATGCAGAAGGCCCCTGATCCAATCGCTGCCTGGTGGTCACAGGCCAACACCAAAGAACGGGACAGATTCGTCGAGTTCATGAGCAACGCTGGCGCCATCAAGGACACCTCCGACGAAGTGCGGAACATTGACGCCGAGATCAGCCAGCTGCTCGAAGAACTGACCGGCTCTGAGCTGGAATCCCTGGATAAGATCACCCTGACCTCTGGCAGAGCCGCCTACAGCTCTCAGACCCTGAGAAACATCACCAACTATATGTACGAGACAGGCTGCGACCTGACCACAGCCAGACAAGAGCTGTACCACGTGGGCAAGAATTGGGCCCCTCCTGCTCCTCCTATCTACGAGCACACAGGCAACCCCAGCGTGGACAGAACCTTCAGCATCATCCACAGATGGCTGTGCAACATGCGGGACCAGTACGGCGAGCCCGAGACAGTGAATATCGAGTACGTCCGCGACGGCTTCAGCAGCACATCTACACAGCTGGCCGAGCAGCGCGAGCGGGATAGAAGATACGCCGACAACCTGAAGATGCTGAGCAACTACGAGGGCGCCAGCAGCAGATCAGATGTGCGGAGAATCAAGGCCCTGCAGAGACAGAACTGCCAGTGCATCTACTGCGGCCGGACCATCACCTTCGAGACATGCCAGATGGACCATGTGCTGCCCCGGAAAGGCCCTGGATCCGATAGCAAGTTCGAGAACCTGGTGGCCACATGCGGCGAGTGCAACAAGTCCAAGAGCGATACCCTGTACATGAACTGGGCCAAGACATACCCCAATACCAACCTGCAGGACGTGCTGAGAAGAATCCAAGAGTGGTCCAAGGACGGCTGGATGACCGACAAAAGATGGCGGCAGTACAAAGAGGCCCTGATCCTGAGACTGGAAGCTACCGAGAAGCAAGAGCCCCTGGACAATCGGAGCATGGAAAGCGTGTCCTACATGGCCAGAGAGCTGCGGAACCGGATCTACGGCTTTTACGGCTGGCACGACCAGGACGACGCCCTGAAACAAGGCAGACAGAGGGTGTTCGTGTCCAGCGGCAGTATGACAGCCGCTGCCAGAAGGACCCCTTTCGAGTCCCCACTGATTAAGGGCGCCGATGAGGAAACCTACGAGAGCAGCCTGCCTTGGCTGGATGGCATGAAGGGCAAGACCAGACTGGATCGGAGACACCATGCCGTGGACGCCAGCATCATTGCCATGATGAGGCCCCAGATCGTGAAGATCCTGACAGAGGCCCAAGAGATCAGAAGCGAGCAGCACGACAAGTACCGGAAGGGCCAGACACCTGACTACGTGTGCAAGCGGCGGGACTACTGGCGGAATTGGAGAGGCACCCCTGACACCAGAGATGAGGAAGTGTTCAACTACTGGGCTGGGGAGCAGCTGAGAACCCTGACCGATCTGGTGTCCCAGAAGATGGCCGACGACGAAATCCCCGTGATCTACCCCACCAGACTGAGACTCGGCAATGGCAGCGCCCACAAGGATACCGTGGTGTCCATGATGACCCGGAAAGTGGGCGACGAGCTGAGCATCACCGCCATCAACAAAGCCGAAAGCGGAGCCCTGTACACAGCCCTGACCAGAGACAGCGACTTCGACTGGAAAACCGGCCTGAGCGCCAATCCTAACCGGCGGATCAGAGTGCACGATAAGTGGTTCGAGGCCGACGATACCATCAAGTTTCTGGAACCTGCCGTGGAAGTGGTGCTGAAGAACAACACCAGAGCCAGAATCGACCCCGAGGCTCTGGATAAGGTGCACAGCACACTGTACGTGCCCGTCAGAGGCGGAATCGCCGAAGCCGGAAATAGCATTCACCACGTGCGGTTCTACAAGATCCCCAAGCTGAACAGCAAGGGCAAGCAGACCGGCAGCATCTACGCCATGCTGAGAGTGCTGACCATCGACCTGGCCATGAACCAGTACGACAAAGAGACAGGCAAGAAGCAGGACCTGTTCACCCTGCCACTGCCTGAAAGCAGCCTGAGCAGAAGATTCAGCGAGCCCAAACTGCGGCAGGCTCTGATCGATGGCACAGCCGAATATCTCGGATGGGCCGTCGTGGACGATGAGCTTGAGATCCCCGCCTTCGCCAACGCCAGAATCACAGAGGAACAGGCCATTAACGGCAGCTTCACCGACAGACTGCTGCACAGCTTTCCCGGCACACACAAGTTCAGATTCGCCGGCTTCTCCCGGAACACCGAGATCGCCATTAGACCTGTGCAGCTGGCCTCTGAGGGCCTGATCGAAACCGATGAGAACCGGAAGAGACAGCAGCTGCGGCTGACCCAGCCTAACACCGAGTACAGCAACAGCATCAAGAACGTGCTGAAGTCCGGCCTGCACCTGAAAGTGAACACCCTGTTCCAGACAGGCATCCTGGTCACCAGGGCCAATAGCCAGGGAAAGCAGAGCATCCGGTTCAGCACAGTGGAAGAGCCCAAGAAGAAGAGGAAGGTG BboCas9/NLS protein sequence (SEQ ID NO: 18)MSQHRRYRIGIDVGLNSVGLAAVEIDANHDNPLDEIPISILNAQSVIHDGGVDPDEAKSATSRRASAGVARRTRRLHKSKRQRLAKLDEVLNELGYPVEDESQFPAGSNPYIAWQVRAKLAETFIPDVETRKRMISIAIRHIARHRGWRNPYSSVADAERMSHIPSPFMVEYAKKLDFEINDRRINGFYHSPWQSVDEEGKRLSKSELEKQPKIEDWNDNPINGKTIAQLVVSSLEPQTKIRRDLTHGLQTESTLNIQTEKLHQSDYIHELETIFERQHVDQTTQEQLLEATFHTKNPKAVGAAAKLVGKDALDSRYYRASRATPAFEEYRVMAAIDTLRIREHGTERQLTTDERRKLFDFIKGLPSKKTKNEPSISSLTWGDVADFLGIQRIDLRGLGSLKDGEPVSAKQPPVIETNDIMQKAPDPIAAWWSQANTKERDRFVEFMSNAGAIKDTSDEVRNIDAEISQLLEELTGSELESLDKITLTSGRAAYSSQTLRNITNYMYETGCDLTTARQELYHVGKNWAPPAPPIYEHIGNPSVDRIFSIIHRWLCNMRDQYGEPETVNIEYVRDGFSSTSTQLAEQRERDRRYADNLKMLSNYEGASSRSDVRRIKALQRQNCQCIYCGRTITFETCQMDHVLPRKGPGSDSKFENLVATCGECNKSKSDTLYMNWAKTYPNTNLQDVLRRIQEWSKDGWMTDKRWRQYKEALILRLEATEKQEPLDNRSMESVSYMARELRNRIYGFYGWHDQDDALKQGRQRVFVSSGSMTAAARRTPFESPLIKGADEETYESSLPWLDGMKGKTRLDRRHHAVDASIIAMMRPQIVKILTEAQEIRSEQHDKYRKGQTPDYVCKRRDYWRNWRGTPDTRDEEVFNYWAGEQLRILTDLVSQKMADDEIPVIYPTRLRLGNGSAHKDTVVSMMTRKVGDELSITAINKAESGALYTALTRDSDFDWKTGLSANPNRRIRVHDKWFEADDTIKFLEPAVEVVLKNNTRARIDPEALDKVHSTLYVPVRGGIAEAGNSIHHVRFYKIPKLNSKGKQTGSIYAMLRVLTIDLAMNQYDKETGKKQDLFTLPLPESSLSRRFSEPKLRQALIDGTAEYLGWAVVDDELEIPAFANARITEEQAINGSFTDRLLHSFPGTHKFRFAGFSRNTEIAIRPVQLASEGLIETDENRKRQQLRLTQPNTEYSNSIKNVLKSGLHLKVNTLFQTGILVTRANSQGKQSIRFSTVEEPKKKRKVAceCas9/NLS DNA sequence (SEQ ID NO: 19)ATGGGCGGATCTGAAGTGGGAACCGTGCCTGTGACTTGGAGACTGGGAGTCGATGTGGGCGAGAGATCCATTGGACTGGCCGCCGTGTCCTACGAAGAGGACAAGCCCAAAGAAATCCTGGCTGCTGTGTCCTGGATTCACGATGGCGGAGTGGGCGACGAAAGAAGCGGAGCTAGTAGACTGGCCCTGAGAGGCATGGCCAGAAGGGCTAGACGGCTGCGGAGATTCCGTAGAGCCAGACTGCGCGACCTGGACATGCTGCTGTCTGAACTCGGATGGACCCCTCTGCCTGACAAGAACGTGTCACCTGTGGATGCCTGGCTGGCCAGAAAGAGACTGGCCGAGGAATACGTGGTGGACGAGACAGAGAGAAGAAGGCTGCTGGGCTACGCCGTGTCTCACATGGCTAGACATAGAGGCTGGCGGAACCCCTGGACCACCATCAAGGACCTGAAGAACCTGCCTCAGCCTAGCGACAGCTGGGAGAGAACCAGAGAAAGCCTGGAAGCCCGGTACTCCGTGTCTCTGGAACCTGGCACAGTTGGACAGTGGGCCGGATACCTGCTGCAGAGAGCCCCTGGCATCAGACTGAACCCTACACAGCAGAGCGCCGGAAGAAGGGCCGAACTGTCTAATGCCACCGCCTTCGAGACAAGACTGCGGCAAGAGGATGTGCTGTGGGAGCTGAGATGTATCGCCGACGTTCAGGGCCTGCCTGAGGACGTGGTGTCCAATGTGATCGACGCCGTGTTCTGCCAGAAAAGACCTAGCGTGCCCGCCGAGAGAATCGGCAGAGATCCTCTCGATCCCAGCCAGCTGAGAGCCAGCAGAGCCTGCCTGGAATTTCAAGAGTACCGGATCGTGGCCGCTGTGGCCAACCTGAGAATCAGAGATGGCAGCGGCAGCAGACCCCTGAGTCTGGAAGAAAGAAACGCCGTGATCGAGGCCCTGCTGGCCCAGACAGAAAGAAGCCTCACTTGGAGCGACATTGCCCTGGAAATCCTGAAGCTGCCCAACGAGAGCGACCTGACCTCTGTGCCTGAAGAGGATGGCCCAAGCAGCCTGGCCTACTCTCAGTTCGCCCCTTTCGATGAGACAAGCGCCCGGATCGCCGAGTTTATCGCCAAGAACAGACGGAAGATCCCCACATTCGCCCAGTGGTGGCAAGAGCAGGATCGGACCAGTAGAAGCGATCTGGTGGCTGCCCTGGCCGACAATTCTATTGCCGGCGAGGAAGAACAAGAGCTGCTGGTGCATCTGCCCGACGCCGAACTTGAAGCTCTGGAAGGACTGGCTCTGCCCTCTGGCAGAGTGGCCTATAGCAGACTGACACTGAGCGGCCTGACCAGAGTGATGAGAGATGATGGCGTGGACGTGCACAACGCCCGCAAGACATGCTTCGGAGTGGACGACAATTGGCGGCCTCCACTGCCTGCTCTGCATGAAGCTACAGGACACCCCGTGGTGGATAGAAACCTGGCTATCCTGCGGAAGTTCCTGAGCAGCGCCACCATGAGATGGGGCCCTCCACAGTCTATCGTGGTGGAACTTGCCAGAGGCGCCAGCGAGAGCAGAGAAAGGCAGGCCGAAGAAGAAGCCGCTCGGAGAGCCCACAGAAAGGCCAACGACAGAATTAGAGCCGAACTCAGAGCCTCCGGCCTGAGCGATCCTTCTCCTGCCGATCTTGTTAGAGCCCGGCTGCTGGAACTGTACGACTGCCACTGTATGTACTGTGGCGCCCCTATCTCCTGGGAGAACAGCGAGCTGGATCACATCGTGCCCAGAACAGATGGCGGATCCAACAGACACGAGAACCTGGCCATTACATGCGGCGCCTGCAACAAAGAAAAAGGCAGAAGGCCCTTCGCCAGCTGGGCCGAGACAAGCAATAGAGTGCAGCTGCGGGACGTGATCGACCGGGTGCAGAAGCTGAAGTACAGCGGCAACATGTACTGGACCCGGGACGAGTTCAGCCGGTACAAGAAAAGCGTGGTGGCCCGGCTGAAGCGGAGAACCTCTGATCCTGAAGTGATCCAGAGCATCGAGAGCACCGGCTATGCTGCCGTGGCTCTGAGAGATAGACTGCTGAGCTACGGCGAGAAGAATGGCGTGGCACAGGTGGCCGTTTTTAGAGGCGGAGTGACAGCCGAGGCCAGAAGATGGCTGGACATCTCCATCGAGCGGCTGTTCAGTAGAGTGGCCATCTTCGCCCAGAGCACCTCCACCAAGAGGCTGGATAGAAGGCACCACGCCGTGGATGCTGTGGTGCTGACAACACTGACACCCGGCGTGGCCAAGACACTGGCTGATGCTAGAAGCAGAAGAGTGTCCGCCGAGTTCTGGCGCAGACCAAGCGACGTGAACAGACACAGCACCGAGGAACCTCAGAGCCCCGCCTACAGACAGTGGAAAGAGAGCTGTTCTGGCCTGGGCGACCTGCTGATTTCTACCGCCGCCAGAGATTCTATCGCCGTGGCTGCTCCTCTGAGACTGAGGCCAACAGGCGCACTGCACGAGGAAACCCTGAGAGCCTTTAGCGAGCACACAGTGGGAGCCGCTTGGAAGGGCGCTGAGCTGAGAAGAATCGTGGAACCCGAAGTGTACGCCGCCTTCCTGGCACTTACAGATCCTGGCGGCAGATTCCTGAAGGTGTCCCCTAGCGAAGATGTGCTGCCTGCCGACGAGAACAGGCACATTGTGCTGAGCGACAGAGTGCTGGGCCCCAGAGACAGAGTGAAACTGTTCCCCGACGACCGGGGCAGCATCAGAGTCAGAGGTGGCGCAGCCTATATCGCCAGCTTTCACCACGCCAGAGTGTTCAGATGGGGAAGCAGCCACTCTCCTAGCTTCGCCCTGCTGAGAGTCTCTCTGGCTGATCTGGCTGTGGCTGGCCTGCTTAGAGATGGGGTCGACGTGTTCACAGCCGAGCTGCCACCTTGGACTCCCGCTTGGAGATATGCCTCTATCGCCCTGGTCAAGGCCGTGGAAAGCGGCGACGCTAAGCAAGTTGGATGGCTGGTGCCTGGCGACGAACTGGATTTTGGACCTGAGGGCGTGACAACCGCTGCCGGCGATCTGAGCATGTTCCTGAAGTACTTTCCCGAGCGGCACTGGGTCGTGACCGGCTTCGAAGATGACAAGAGGATCAACCTGAAGCCTGCCTTCCTGTCTGCCGAACAGGCTGAGGTGCTGAGGACTGAGAGAAGCGACAGACCCGACACACTGACAGAGGCCGGCGAAATTCTGGCCCAGTTCTTCCCTAGATGTTGGCGGGCCACAGTGGCTAAGGTGCTGTGCCATCCTGGCCTGACCGTGATCAGAAGAACAGCCCTGGGACAGCCTAGGTGGCGGAGAGGACATCTGCCTTATTCATGGCGGCCTTGGAGCGCCGATCCTTGGAGTGGCGGAACACCTCCCAAGAAGAAGAGGAAGGTGAceCas9/NLS protein sequence (SEQ ID NO: 20)MGGSEVGTVPVTWRLGVDVGERSIGLAAVSYEEDKPKEILAAVSWIHDGGVGDERSGASRLALRGMARRARRLRRFRRARLRDLDMLLSELGWTPLPDKNVSPVDAWLARKRLAEEYVVDETERRRLLGYAVSHMARHRGWRNPWTTIKDLKNLPQPSDSWERTRESLEARYSVSLEPGTVGQWAGYLLQRAPGIRLNPTQQSAGRRAELSNATAFETRLRQEDVLWELRCIADVQGLPEDVVSNVIDAVFCQKRPSVPAERIGRDPLDPSQLRASRACLEFQEYRIVAAVANLRIRDGSGSRPLSLEERNAVIEALLAQTERSLTWSDIALEILKLPNESDLTSVPEEDGPSSLAYSQFAPFDETSARIAEFIAKNRRKIPTFAQWWQEQDRTSRSDLVAALADNSIAGEEEQELLVHLPDAELEALEGLALPSGRVAYSRLTLSGLTRVMRDDGVDVHNARKTCFGVDDNWRPPLPALHEATGHPVVDRNLAILRKFLSSATMRWGPPQSIVVELARGASESRERQAEEEAARRAHRKANDRIRAELRASGLSDPSPADLVRARLLELYDCHCMYCGAPISWENSELDHIVPRTDGGSNRHENLAITCGACNKEKGRRPFASWAETSNRVQLRDVIDRVQKLKYSGNMYWTRDEFSRYKKSVVARLKRRTSDPEVIQSIESTGYAAVALRDRLLSYGEKNGVAQVAVFRGGVTAEARRWLDISIERLFSRVAIFAQSTSTKRLDRRHHAVDAVVLTTLTPGVAKTLADARSRRVSAEFWRRPSDVNRHSTEEPQSPAYRQWKESCSGLGDLLISTAARDSIAVAAPLRLRPTGALHEETLRAFSEHTVGAAWKGAELRRIVEPEVYAAFLALTDPGGRFLKVSPSEDVLPADENRHIVLSDRVLGPRDRVKLFPDDRGSIRVRGGAAYIASFHHARVFRWGSSHSPSFALLRVSLADLAVAGLLRDGVDVFTAELPPWTPAWRYASIALVKAVESGDAKQVGWLVPGDELDFGPEGVTTAAGDLSMFLKYFPERHWVVTGFEDDKRINLKPAFLSAEQAEVLRTERSDRPDTLTEAGEILAQFFPRCWRATVAKVLCHPGLTVIRRTALGQPRWRRGHLPYSWRPWSADPWSGGTPPKKKRKV AheCas9/NLS DNA sequence (SEQ ID NO: 21)ATGGCCTATAGACTGGGCCTCGACATCGGCATCACATCTGTTGGATGGGCCGTCGTGGCCCTGGAAAAGGATGAGTCTGGACTGAAGCCCGTGCGCATCCAGGATCTGGGCGTCAGAATCTTCGACAAGGCCGAGGATAGCAAGACCGGCGCTTCTCTGGCTCTGCCCAGAAGAGAAGCCAGAAGCGCCAGAAGAAGAACCCGGCGGAGAAGGCACAGACTGTGGCGCGTGAAAAGACTGCTGGAACAGCACGGCATCCTGAGCATGGAACAGATCGAGGCCCTGTACGCCCAGAGAACAAGCAGCCCTGATGTGTATGCCCTGAGAGTGGCCGGCCTGGACAGATGTCTGATCGCCGAAGAGATCGCCCGGGTGCTGATTCACATTGCCCACAGAAGAGGCTTCCAGAGCAACAGAAAGAGCGAGATCAAGGACAGCGACGCCGGCAAGCTGCTGAAGGCCGTGCAAGAGAACGAGAACCTGATGCAGAGCAAGGGCTACAGAACCGTGGCCGAGATGCTGGTGTCTGAGGCCACAAAGACAGACGCCGAGGGAAAGCTGGTGCACGGCAAGAAGCACGGCTACGTCAGCAACGTGCGGAACAAGGCCGGCGAGTACAGACACACAGTGTCCAGACAGGCCATCGTGGACGAAGTGCGGAAGATTTTCGCCGCTCAGAGAGCCCTGGGCAACGACGTGATGAGCGAGGAACTGGAAGATAGCTACCTGAAGATCCTGTGCAGCCAGCGGAACTTCGATGATGGCCCTGGCGGCGATTCTCCTTATGGACACGGAAGCGTTAGCCCCGACGGCGTCAGACAGAGCATCTACGAGAGAATGGTCGGAAGCTGCACCTTCGAGACAGGCGAGAAGAGAGCCCCTAGAAGCAGCTACAGCTTCGAGCGGTTTCAGCTGCTGACCAAGGTGGTCAACCTGCGGATCTACCGGCAGCAAGAGGATGGCGGCAGATACCCTTGTGAACTGACCCAGACCGAGCGGGCCAGAGTGATCGATTGTGCCTACGAGCAGACCAAGATCACCTACGGAAAGCTGAGAAAGCTGCTGGACATGAAGGACACCGAGAGCTTTGCCGGCCTGACCTACGGCCTGAACAGAAGCAGAAACAAGACCGAGGACACCGTGTTCGTGGAAATGAAGTTCTACCACGAAGTCCGCAAGGCCCTGCAGAGAGCCGGGGTTTTCATTCAGGACCTGAGCATCGAGACACTGGACCAGATCGGCTGGATTCTGAGCGTGTGGAAGTCCGACGACAACCGGCGGAAGAAGCTGTCTACACTGGGCCTGAGCGACAACGTGATCGAAGAACTGCTGCCCCTGAACGGCTCCAAGTTTGGCCACCTGAGCCTGAAGGCCATCAGAAAGATCCTGCCTTTCCTGGAAGATGGGTACAGCTACGACGTGGCCTGTGAACTGGCCGGCTATCAGTTTCAGGGCAAGACAGAGTACGTGAAGCAGCGGCTGCTGCCTCCACTTGGAGAAGGCGAAGTGACAAACCCCGTTGTGCGCAGAGCACTGAGCCAGGCCATCAAGGTTGTGAACGCCGTGATCAGAAAGCACGGCAGCCCAGAGAGCATCCACATCGAACTGGCCAGAGAGCTGAGCAAGAACCTGGACGAGCGGAGAAAGATCGAGAAGGCCCAGAAAGAAAATCAGAAGAACAACGAGCAGATTAAGGACGAGATCCGCGAGATCCTGGGATCCGCCCATGTGACCGGAAGAGACATCGTGAAGTACAAGCTGTTCAAACAGCAACAAGAGTTCTGCATGTACAGCGGCGAGAAGCTGGACGTGACCAGACTGTTTGAGCCTGGCTATGCCGAGGTGGACCACATCATCCCTTACGGCATCAGCTTCGACGACTCCTACGACAACAAGGTGCTGGTTAAGACCGAGCAGAACCGGCAGAAGGGCAATAGAACCCCTCTGGAATACCTGCGGGACAAGCCTGAGCAGAAGGCCAAGTTTATCGCCCTGGTGGAATCTATCCCTCTGAGCCAGAAAAAGAAAAACCACCTCCTGATGGACAAGCGGGCCATCGACCTGGAACAAGAGGGCTTCAGAGAGCGGAACCTGAGCGATACCCGGTACATCACACGGGCCCTGATGAACCACATCCAGGCTTGGCTGCTGTTCGACGAGACAGCCAGCACCAGATCCAAGAGGGTCGTGTGTGTGAATGGCGCCGTGACCGCCTACATGAGAGCTAGATGGGGCCTGACAAAGGATAGAGATGCCGGCGATAAGCACCACGCCGCTGATGCTGTGGTGGTGGCCTGTATCGGAGACAGCCTGATCCAGAGAGTGACCAAATACGACAAGTTCAAGCGGAACGCCCTGGCCGACCGGAACAGATATGTGCAGCAGGTTTCCAAGAGCGAGGGCATCACCCAGTACGTGGACAAAGAAACCGGCGAGGTGTTCACCTGGGAGTCCTTCGATGAGCGGAAGTTCCTGCCTAACGAGCCCCTGGAACCTTGGCCATTCTTCAGGGATGAGCTGCTGGCCAGACTGAGCGACGACCCCTCCAAGAACATCAGAGCCATCGGCCTGCTGACCTACAGCGAGACTGAGCAGATCGATCCCATCTTCGTGTCCAGAATGCCCACCAGAAAAGTGACCGGCGCAGCCCACAAAGAGACAATCAGATCCCCACGGATCGTGAAGGTGGACGATAACAAGGGCACCGAGATCCAGGTGGTGGTGTCTAAGGTGGCCCTGACCGAGCTGAAGCTGACCAAAGACGGCGAAATCAAGGATTACTTCAGGCCCGAGGACGACCCCAGACTGTACAACACCCTGAGAGAACGGCTGGTGCAGTTCGGCGGAGATGCCAAGGCCGCCTTCAAAGAACCCGTGTACAAGATCAGCAAGGACGGCTCTGTGCGGACCCCTGTGCGGAAAGTGAAGATTCAAGAGAAGCTGACACTGGGCGTGCCAGTGCATGGCGGAAGAGGAATTGCCGAGAATGGCGGCATGGTCCGAATCGACGTGTTCGCCAAAGGCGGCAAGTACTACTTCGTGCCCATCTACGTGGCCGACGTGCTGAAGAGAGAGCTGCCCAACAGACTGGCCACCGCTCACAAGCCTTACAGCGAATGGCGCGTGGTGGACGACAGCTACCAGTTCAAGTTCTCTCTGTACCCCAACGATGCCGTGATGATCAAGCCCAGCAGAGAGGTGGACATCACCTACAAGGACCGGAAAGAGCCCGTCGGCTGCCGGATCATGTACTTTGTGTCCGCCAATATCGCCAGCGCCTCCATCAGCCTGAGAACCCACGATAACTCCGGCGAGCTGGAAGGACTGGGCATCCAAGGACTGGAAGTGTTCGAGAAATACGTCGTGGGCCCTCTGGGCGACACACACCCTGTGTACAAAGAACGGCGGATGCCCTTCAGAGTGGAACGGAAGATGAACCCCAAGAAGAAGAGGAAGGTGAheCas9/NLS protein sequence (SEQ ID NO: 22)MAYRLGLDIGITSVGWAVVALEKDESGLKPVRIQDLGVRIFDKAEDSKTGASLALPRREARSARRRTRRRRHRLWRVKRLLEQHGILSMEQIEALYAQRTSSPDVYALRVAGLDRCLIAEEIARVLIHIAHRRGFQSNRKSEIKDSDAGKLLKAVQENENLMQSKGYRTVAEMLVSEATKTDAEGKLVHGKKHGYVSNVRNKAGEYRHTVSRQAIVDEVRKIFAAQRALGNDVMSEELEDSYLKILCSQRNFDDGPGGDSPYGHGSVSPDGVRQSIYERMVGSCTFETGEKRAPRSSYSFERFQLLTKVVNLRIYRQQEDGGRYPCELTQTERARVIDCAYEQTKITYGKLRKLLDMKDTESFAGLTYGLNRSRNKTEDTVFVEMKFYHEVRKALQRAGVFIQDLSIETLDQIGWILSVWKSDDNRRKKLSTLGLSDNVIEELLPLNGSKFGHLSLKAIRKILPFLEDGYSYDVACELAGYQFQGKTEYVKQRLLPPLGEGEVTNPVVRRALSQAIKVVNAVIRKHGSPESIHIELARELSKNLDERRKIEKAQKENQKNNEQIKDEIREILGSAHVTGRDIVKYKLFKQQQEFCMYSGEKLDVTRLFEPGYAEVDHIIPYGISFDDSYDNKVLVKTEQNRQKGNRTPLEYLRDKPEQKAKFIALVESIPLSQKKKNHLLMDKRAIDLEQEGFRERNLSDTRYITRALMNHIQAWLLFDETASTRSKRVVCVNGAVTAYMRARWGLTKDRDAGDKHHAADAVVVACIGDSLIQRVTKYDKFKRNALADRNRYVQQVSKSEGITQYVDKETGEVFTWESFDERKFLPNEPLEPWPFFRDELLARLSDDPSKNIRAIGLLTYSETEQIDPIFVSRMPTRKVTGAAHKETIRSPRIVKVDDNKGTEIQVVVSKVALTELKLTKDGEIKDYFRPEDDPRLYNTLRERLVQFGGDAKAAFKEPVYKISKDGSVRTPVRKVKIQEKLTLGVPVHGGRGIAENGGMVRIDVFAKGGKYYFVPIYVADVLKRELPNRLATAHKPYSEWRVVDDSYQFKFSLYPNDAVMIKPSREVDITYKDRKEPVGCRIMYFVSANIASASISLRTHDNSGELEGLGIQGLEVFEKYVVGPLGDTHPVYKERRMPFRVERKMNPKKKRKV WsuCas9/NLS DNA sequence (SEQ ID NO: 23)ATGCTGGTGTCCCCTATCTCTGTGGATCTCGGCGGCAAGAATACCGGCTTCTTCAGCTTCACCGACAGCCTGGACAATAGCCAGAGCGGCACCGTGATCTACGACGAGAGCTTCGTGCTGAGCCAAGTGGGCAGAAGAAGCAAGCGGCACAGCAAGCGGAACAACCTGAGAAACAAGCTGGTCAAGCGGCTGTTCCTGCTGATCCTGCAAGAGCACCACGGCCTGAGCATCGACGTTCTGCCCGATGAGATCCGGGGCCTGTTCAACAAGAGAGGCTACACCTACGCCGGCTTCGAGCTGGACGAGAAGAAGAAGGACGCCCTGGAAAGCGATACCCTGAAAGAGTTCCTGAGCGAGAAGCTGCAGTCCATCGACAGAGACAGCGACGTGGAAGATTTCCTGAACCAGATCGCCAGCAACGCCGAGAGCTTTAAGGACTACAAGAAAGGCTTCGAGGCCGTGTTCGCCAGCGCCACACACAGCCCCAACAAGAAGCTGGAACTGAAGGACGAGCTGAAGTCCGAGTACGGCGAGAACGCCAAAGAACTGCTGGCCGGCCTGAGAGTGACCAAAGAGATCCTGGACGAGTTCGACAAGCAAGAGAACCAGGGCAACCTGCCTCGGGCCAAGTACTTTGAGGAACTGGGCGAGTATATCGCCACCAACGAGAAAGTCAAGAGCTTCTTCGACAGCAACAGCCTGAAGCTGACCGACATGACCAAGCTGATCGGCAACATCAGCAACTACCAGCTGAAAGAGCTGCGGCGGTACTTCAACGACAAAGAGATGGAAAAGGGCGACATCTGGATTCCCAACAAGCTGCACAAGATCACCGAGAGATTTGTGCGGAGCTGGCACCCCAAGAACGACGCCGATAGACAGAGAAGGGCCGAGCTGATGAAGGACCTGAAGTCCAAAGAAATCATGGAACTGCTGACCACCACCGAGCCTGTGATGACAATCCCTCCTTACGACGACATGAACAACAGAGGCGCCGTGAAGTGTCAGACCCTGCGGCTGAATGAGGAATACCTGGACAAACATCTGCCCAACTGGCGGGATATCGCCAAGAGACTGAACCACGGCAAGTTCAACGACGACCTGGCCGACTCTACCGTGAAGGGCTACAGCGAGGATAGCACCCTGCTGCACAGACTGCTGGACACCTCTAAAGAGATCGACATCTACGAGCTGCGGGGCAAGAAGCCCAACGAGCTGCTGGTTAAGACACTGGGCCAGAGCGACGCCAACAGACTGTATGGCTTCGCCCAGAACTACTATGAGCTGATCCGGCAGAAAGTGCGCGCTGGCATTTGGGTGCCCGTGAAGAACAAGGATGACTCCCTGAACCTGGAAGATAACTCCAACATGCTGAAGCGGTGCAACCACAATCCTCCACACAAGAAGAATCAGATCCACAACCTGGTGGCCGGCATCCTGGGAGTGAAACTGGATGAGGCCAAGTTCGCCGAGTTCGAGAAAGAGCTTTGGAGCGCCAAAGTGGGCAACAAGAAACTGAGCGCCTACTGCAAGAACATCGAGGAACTGAGAAAGACCCACGGCAACACCTTCAAGATCGATATAGAGGAACTGCGCAAGAAGGACCCCGCCGAGCTGTCCAAAGAGGAAAAGGCCAAGCTGAGACTGACCGACGACGTGATCCTGAATGAGTGGTCCCAGAAGATCGCCAACTTCTTTGACATCGACGACAAGCACCGGCAGCGGTTCAACAACCTGTTCAGCATGGCCCAGCTGCACACAGTGATCGACACACCCAGAAGCGGCTTCAGCTCTACCTGCAAAAGATGCACCGCCGAGAACAGGTTCAGAAGCGAGACAGCCTTCTACAACGACGAGACAGGCGAGTTCCACAAGAAGGCCACAGCCACCTGTCAGAGACTGCCCGCTGATACCCAGAGGCCTTTCAGCGGAAAGATCGAGCGGTACATCGACAAGCTGGGATACGAGCTGGCCAAGATCAAGGCTAAAGAACTGGAAGGCATGGAAGCTAAAGAAATCAAGGTGCCCATCATCCTGGAACAGAACGCCTTCGAGTACGAGGAAAGCCTGCGGAAGTCTAAGACCGGATCCAACGACAGAGTGATCAACTCCAAGAAAGACCGCGACGGAAAGAAACTGGCCAAGGCCAAAGAGAACGCCGAGGACAGGCTGAAGGACAAGGACAAGCGGATCAAGGCCTTCAGCAGCGGCATCTGCCCTTACTGCGGAGATACCATCGGAGATGACGGCGAGATCGACCACATCCTGCCTAGAAGCCACACACTGAAAATCTACGGGACCGTGTTCAACCCCGAGGGCAATCTGATCTACGTGCACCAGAAGTGCAACCAGGCCAAAGCCGACAGCATCTACAAGCTGAGCGATATCAAGGCCGGCGTGTCAGCCCAGTGGATTGAAGAACAGGTGGCCAACATTAAGGGGTACAAGACCTTCAGCGTGCTGTCCGCCGAACAGCAGAAGGCCTTTAGATACGCCCTGTTCCTCCAGAACGACAACGAGGCCTACAAAAAGGTGGTGGACTGGCTGCGGACCGACCAGTCTGCTAGAGTGAACGGCACACAGAAGTACCTGGCCAAAAAGATCCAAGAGAAGCTCACCAAGATGCTGCCTAACAAGCACCTGAGCTTCGAGTTCATCCTGGCCGATGCCACCGAGGTGTCAGAGCTGAGAAGGCAGTACGCCAGACAGAACCCTCTGCTGGCTAAGGCCGAGAAGCAGGCCCCTTCTTCTCACGCCATTGATGCCGTGATGGCCTTCGTGGCCAGATACCAGAAGGTGTTCAAGGACGGCACCCCTCCTAACGCCGATGAGGTGGCAAAACTGGCTATGCTGGACAGCTGGAACCCCGCCTCTAATGAGCCTCTGACAAAGGGCCTGTCCACGAACCAGAAAATCGAGAAGATGATCAAGAGCGGCGACTACGGCCAGAAAAACATGAGAGAGGTGTTCGGCAAGTCCATCTTCGGAGAGAATGCCATCGGCGAGAGATACAAGCCCATCGTGGTTCAAGAAGGCGGCTACTACATCGGCTACCCCGCCACAGTGAAAAAGGGCTACGAACTGAAGAACTGCAAGGTGGTCACCAGCAAGAACGATATTGCCAAGCTGGAAAAGATCATCAAGAACCAGGACCTGATCTCTCTGAAAGAGAATCAGTACATCAAAATCTTCTCCATCAACAAGCAGACCATCAGCGAGCTGAGCAACCGCTACTTCAACATGAATTACAAGAACCTGGTCGAGCGGGACAAAGAAATTGTGGGACTGCTTGAGTTTATCGTCGAGAACTGCCGGTACTACACCAAGAAAGTGGACGTGAAGTTCGCCCCTAAGTACATCCACGAGACAAAGTACCCCTTCTACGATGACTGGCGGAGATTCGACGAGGCCTGGCGGTATCTGCAAGAAAACCAGAACAAGACCAGCTCCAAGGACCGCTTCGTGATCGATAAGAGCAGCCTGAACGAGTACTACCAGCCAGACAAGAATGAGTACAAGCTGGACGTGGACACCCAGCCTATCTGGGACGACTTCTGCCGGTGGTACTTCCTGGACAGATACAAGACCGCCAACGACAAGAAGTCCATCCGCATCAAGGCCCGCAAGACATTCTCCCTGCTGGCTGAGTCTGGCGTGCAGGGCAAAGTGTTCCGGGCCAAGAGAAAGATCCCTACCGGCTACGCCTATCAGGCCCTGCCTATGGACAACAACGTGATCGCTGGCGATTACGCCAACATTCTGCTGGAAGCCAACAGCAAGACCCTGAGCCTGGTGCCTAAGAGCGGCATCAGCATTGAGAAGCAGCTGGACAAAAAGCTCGACGTCATCAAAAAGACCGACGTGCGCGGCCTGGCAATCGACAACAACTCCTTCTTCAACGCCGACTTCGACACACACGGCATCCGGCTGATCGTGGAAAACACCAGCGTGAAAGTGGGAAACTTCCCCATCAGCGCCATCGATAAGTCCGCCAAGCGGATGATCTTCAGAGCCCTGTTTGAGAAAGAGAAGGGGAAGCGCAAGAAAAAGACCACCATCAGCTTCAAAGAAAGCGGCCCTGTGCAGGACTACCTCAAGGTGTTCCTGAAAAAGATCGTGAAGATCCAGCTGAGAACCGACGGCTCCATCTCCAACATCGTCGTGCGGAAGAATGCCGCCGATTTCACCCTGAGCTTTAGAAGCGAGCACATCCAGAAACTGCTGAAGCCCAAGAAGAAGAGGAAGGTG WsuCas9/NLS protein sequence (SEQ ID NO: 24)MLVSPISVDLGGKNTGFFSFTDSLDNSQSGTVIYDESFVLSQVGRRSKRHSKRNNLRNKLVKRLFLLILQEHHGLSIDVLPDEIRGLFNKRGYTYAGFELDEKKKDALESDTLKEFLSEKLQSIDRDSDVEDFLNQIASNAESFKDYKKGFEAVFASATHSPNKKLELKDELKSEYGENAKELLAGLRVTKEILDEFDKQENQGNLPRAKYFEELGEYIATNEKVKSFFDSNSLKLTDMTKLIGNISNYQLKELRRYFNDKEMEKGDIWIPNKLHKITERFVRSWHPKNDADRQRRAELMKDLKSKEIMELLTTTEPVMTIPPYDDMNNRGAVKCQTLRLNEEYLDKHLPNWRDIAKRLNHGKFNDDLADSTVKGYSEDSTLLHRLLDTSKEIDIYELRGKKPNELLVKTLGQSDANRLYGFAQNYYELIRQKVRAGIWVPVKNKDDSLNLEDNSNMLKRCNHNPPHKKNQIHNLVAGILGVKLDEAKFAEFEKELWSAKVGNKKLSAYCKNIEELRKTHGNTFKIDIEELRKKDPAELSKEEKAKLRLTDDVILNEWSQKIANFFDIDDKHRQRFNNLFSMAQLHTVIDTPRSGFSSTCKRCTAENRFRSETAFYNDETGEFHKKATATCQRLPADTQRPFSGKIERYIDKLGYELAKIKAKELEGMEAKEIKVPIILEQNAFEYEESLRKSKTGSNDRVINSKKDRDGKKLAKAKENAEDRLKDKDKRIKAFSSGICPYCGDTIGDDGEIDHILPRSHTLKIYGTVFNPEGNLIYVHQKCNQAKADSIYKLSDIKAGVSAQWIEEQVANIKGYKTFSVLSAEQQKAFRYALFLQNDNEAYKKVVDWLRTDQSARVNGTQKYLAKKIQEKLTKMLPNKHLSFEFILADATEVSELRRQYARQNPLLAKAEKQAPSSHAIDAVMAFVARYQKVFKDGTPPNADEVAKLAMLDSWNPASNEPLTKGLSTNQKIEKMIKSGDYGQKNMREVFGKSIFGENAIGERYKPIVVQEGGYYIGYPATVKKGYELKNCKVVTSKNDIAKLEKIIKNQDLISLKENQYIKIFSINKQTISELSNRYFNMNYKNLVERDKEIVGLLEFIVENCRYYTKKVDVKFAPKYIHETKYPFYDDWRRFDEAWRYLQENQNKTSSKDRFVIDKSSLNEYYQPDKNEYKLDVDTQPIWDDFCRWYFLDRYKTANDKKSIRIKARKTFSLLAESGVQGKVFRAKRKIPTGYAYQALPMDNNVIAGDYANILLEANSKTLSLVPKSGISIEKQLDKKLDVIKKTDVRGLAIDNNSFFNADFDTHGIRLIVENTSVKVGNFPISAIDKSAKRMIFRALFEKEKGKRKKKTTISFKESGPVQDYLKVFLKKIVKIQLRTDGSISNIVVRKNAADFTLSFRSEHIQKLLKPKKKRKV NsaCas9/NLS DNA sequence (SEQ ID NO: 25)ATGAAGAAGATCCTGGGCGTCGACCTGGGCATCACCAGCTTTGGATACGCCATCCTGCAAGAGACAGGCAAGGACCTGTACAGATGCCTGGACAACAGCGTGGTCATGCGGAACAACCCCTACGACGAGAAGTCTGGCGAGAGCAGCCAGAGCATCCGCAGCACCCAGAAATCCATGCGGCGGCTGATCGAGAAGCGGAAGAAACGGATCAGATGCGTGGCCCAGACAATGGAACGCTACGGCATCCTGGACTACTCCGAGACAATGAAGATCAACGACCCCAAGAACAACCCGATCAAGAACAGATGGCAGCTGAGAGCCGTGGACGCCTGGAAAAGACCTCTGAGCCCTCAAGAGCTGTTCGCCATCTTTGCCCACATGGCCAAGCACCGGGGCTACAAGTCTATCGCCACCGAGGACCTGATCTACGAGCTGGAACTGGAACTCGGCCTGAACGACCCTGAGAAAGAGTCCGAGAAGAAGGCCGACGAGCGGAGACAGGTGTACAACGCCCTGAGACACCTGGAAGAACTGCGGAAGAAGTACGGCGGCGAGACAATCGCCCAGACCATCCACAGAGCTGTGGAAGCCGGCGACCTGCGGAGCTACAGAAACCACGACGACTACGAGAAGATGATCCGCAGAGAGGACATCGAGGAAGAGATTGAGAAGGTCCTGCTGCGGCAGGCTGAACTGGGAGCACTTGGACTGCCTGAGGAACAGGTGTCCGAGCTGATCGATGAGCTGAAGGCCTGCATCACCGACCAAGAGATGCCCACCATCGACGAGAGCCTGTTCGGCAAGTGCACCTTCTACAAGGACGAGCTGGCCGCTCCTGCCTACAGCTACCTGTACGACCTGTACCGGCTGTACAAGAAGCTGGCCGACCTGAACATCGACGGCTACGAAGTGACCCAAGAGGACCGCGAGAAAGTGATCGAGTGGGTCGAGAAAAAGATCGCCCAGGGCAAGAACCTGAAGAAAATCACCCACAAGGACCTCCGGAAGATCCTCGGACTGGCCCCTGAGCAGAAGATTTTCGGCGTCGAGGACGAGAGAATCGTCAAGGGAAAGAAAGAACCCCGGACCTTCGTGCCCTTCTTCTTCCTGGCCGATATCGCCAAGTTCAAAGAACTGTTTGCCAGCATCCAGAAGCACCCCGACGCTCTGCAGATTTTCAGAGAACTGGCCGAGATCCTGCAGCGGAGCAAGACACCTCAAGAGGCCCTGGATAGACTGAGAGCCCTGATGGCCGGCAAGGGCATCGACACCGATGACAGAGAGCTGCTGGAACTCTTCAAGAACAAGCGGAGCGGCACAAGAGAGCTGAGCCACCGCTATATCCTGGAAGCCCTGCCTCTGTTCCTGGAAGGCTATGACGAGAAAGAGGTGCAGAGAATCCTGGGCTTTGACGACCGCGAGGACTACAGCAGATACCCCAAGAGCCTGCGGCATCTGCACCTGAGAGAGGGCAACCTGTTCGAGAAAGAAGAGAATCCCATCAACAACCACGCCGTGAAGTCCCTGGCTTCTTGGGCCCTGGGACTGATCGCTGACCTGTCTTGGAGATACGGCCCCTTCGATGAGATCATCCTGGAAACCACCAGGGACGCCCTGCCTGAGAAGATCCGGAAAGAAATCGACAAGGCCATGCGCGAGAGAGAGAAAGCCCTGGACAAGATCATCGGCAAGTACAAGAAAGAGTTCCCCAGCATCGACAAGCGGCTGGCCAGAAAGATTCAGCTGTGGGAGAGACAGAAAGGCCTCGATCTGTACTCCGGCAAAGTGATCAACCTGAGCCAGCTGCTCGATGGATCCGCCGACATCGAGCACATCGTGCCTCAGTCTCTCGGCGGCCTGAGCACCGACTACAATACCATCGTGACCCTGAAGTCCGTGAACGCCGCCAAGGGCAATAGACTGCCTGGCGATTGGCTGGCCGGAAATCCCGACTACAGAGAACGGATCGGCATGCTGTCTGAGAAGGGCCTGATCGACTGGAAGAAGAGGAAGAACCTGCTGGCCCAGAGCCTGGACGAAATCTACACCGAGAACACCCACAGCAAAGGCATCCGGGCCACAAGCTACCTGGAAGCTCTGGTTGCCCAGGTGCTGAAGCGGTACTACCCATTTCCTGATCCTGAGCTGCGCAAGAATGGCATCGGCGTGCGGATGATCCCCGGAAAAGTGACCAGCAAGACCAGAAGCCTGCTGGGAATCAAGAGCAAGAGCCGCGAGACAAACTTCCACCACGCCGAGGATGCCCTGATTCTGAGCACACTGACCAGAGGCTGGCAGAACCGGCTGCACAGAATGCTGAGAGACAACTACGGCAAGAGCGAGGCCGAGCTGAAAGAACTCTGGAAAAAGTACATGCCCCACATCGAGGGCCTGACACTGGCCGACTATATCGATGAGGCCTTCCGGCGGTTCATGAGCAAGGGCGAAGAGTCCCTGTTCTACCGGGACATGTTCGACACCATCCGGTCCATCAGCTACTGGGTCGACAAGAAGCCTCTGAGCGCCAGCAGCCACAAAGAAACCGTGTACAGCAGCAGACACGAGGTGCCCACACTGAGGAAAAACATTCTGGAAGCCTTCGACAGCCTGAACGTGATCAAGGACCGGCACAAGCTGACCACCGAAGAGTTCATGAAGCGCTACGACAAAGAGATCCGGCAGAAGCTGTGGCTGCACCGCATCGGCAACACCAACGACGAGTCTTACCGCGCCGTGGAAGAGAGAGCCACACAGATTGCCCAGATCCTGACCAGATACCAGCTCATGGACGCCCAGAATGACAAAGAAATTGATGAGAAGTTTCAGCAGGCCCTGAAAGAGCTGATCACAAGCCCCATCGAAGTGACTGGCAAGCTGCTGCGGAAAATGAGATTCGTGTACGACAAGCTGAACGCCATGCAGATCGACAGAGGCCTGGTGGAAACCGACAAGAACATGCTGGGCATCCACATCAGCAAGGGCCCCAATGAGAAGCTGATCTTCAGACGGATGGACGTGAACAACGCCCACGAGCTGCAAAAAGAACGCAGCGGAATCCTGTGCTACCTGAACGAGATGCTGTTCATCTTCAACAAGAAGGGGCTGATTCACTACGGCTGCCTGCGGTCTTACCTCGAAAAAGGCCAGGGCAGCAAGTATATCGCCCTGTTCAACCCTCGGTTCCCCGCCAATCCTAAGGCTCAGCCTAGCAAGTTCACCAGCGACAGCAAGATCAAGCAAGTCGGCATCGGCAGCGCCACCGGAATCATTAAGGCCCACCTGGATCTGGATGGCCACGTGCGCTCTTATGAGGTGTTCGGAACACTGCCCGAGGGCAGCATCGAGTGGTTCAAAGAGGAAAGCGGCTACGGCAGAGTGGAAGATGACCCTCACCACCCCAAGAAGAAGAGGAAGGTG NsaCas9/NLS protein sequence (SEQ ID NO: 26)MKKILGVDLGITSFGYAILQETGKDLYRCLDNSVVMRNNPYDEKSGESSQSIRSTQKSMRRLIEKRKKRIRCVAQTMERYGILDYSETMKINDPKNNPIKNRWQLRAVDAWKRPLSPQELFAIFAHMAKHRGYKSIATEDLIYELELELGLNDPEKESEKKADERRQVYNALRHLEELRKKYGGETIAQTIHRAVEAGDLRSYRNHDDYEKMIRREDIEEEIEKVLLRQAELGALGLPEEQVSELIDELKACITDQEMPTIDESLFGKCTFYKDELAAPAYSYLYDLYRLYKKLADLNIDGYEVTQEDREKVIEWVEKKIAQGKNLKKITHKDLRKILGLAPEQKIFGVEDERIVKGKKEPRTFVPFFFLADIAKFKELFASIQKHPDALQIFRELAEILQRSKTPQEALDRLRALMAGKGIDTDDRELLELFKNKRSGTRELSHRYILEALPLFLEGYDEKEVQRILGFDDREDYSRYPKSLRHLHLREGNLFEKEENPINNHAVKSLASWALGLIADLSWRYGPFDEIILETTRDALPEKIRKEIDKAMREREKALDKIIGKYKKEFPSIDKRLARKIQLWERQKGLDLYSGKVINLSQLLDGSADIEHIVPQSLGGLSTDYNTIVTLKSVNAAKGNRLPGDWLAGNPDYRERIGMLSEKGLIDWKKRKNLLAQSLDEIYTENTHSKGIRATSYLEALVAQVLKRYYPFPDPELRKNGIGVRMIPGKVTSKTRSLLGIKSKSRETNFHHAEDALILSTLTRGWQNRLHRMLRDNYGKSEAELKELWKKYMPHIEGLTLADYIDEAFRRFMSKGEESLFYRDMFDTIRSISYWVDKKPLSASSHKETVYSSRHEVPTLRKNILEAFDSLNVIKDRHKLTTEEFMKRYDKEIRQKLWLHRIGNTNDESYRAVEERATQIAQILTRYQLMDAQNDKEIDEKFQQALKELITSPIEVTGKLLRKMRFVYDKLNAMQIDRGLVETDKNMLGIHISKGPNEKLIFRRMDVNNAHELQKERSGILCYLNEMLFIFNKKGLIHYGCLRSYLEKGQGSKYIALFNPRFPANPKAQPSKFTSDSKIKQVGIGSATGIIKAHLDLDGHVRSYEVFGTLPEGSIEWFKEESGYGRVEDDPHHPK KKRKVRsyCas9/NLS DNA sequence (SEQ ID NO: 27)ATGGCCGAGAAGCAGCACAGATGGGGACTCGACATCGGCACCAATTCTATCGGCTGGGCCGTGATCGCCCTGATCGAAGGCAGACCTGCTGGACTGGTGGCTACCGGCAGCAGAATCTTTAGCGACGGCAGAAACCCCAAGGACGGCAGCTCTCTGGCCGTCGAGAGAAGAGGACCTCGGCAGATGCGGCGGAGAAGAGACAGATATCTCCGGCGGAGGGACAGATTCATGCAGGCCCTGATCAACGTGGGCCTGATGCCTGGGGATGCCGCCGCTAGAAAAGCCCTGGTCACCGAGAATCCCTACGTGCTGAGACAGAGAGGCCTGGACCAAGCTCTGACCCTGCCTGAATTTGGCAGAGCCCTGTTCCACCTGAACCAGCGGAGAGGCTTCCAGAGCAACAGAAAGACCGATCGGGCCACCGCCAAAGAAAGCGGCAAAGTGAAGAACGCCATTGCCGCCTTCAGAGCCGGCATGGGCAATGCCAGAACAGTGGGAGAAGCCCTGGCCAGACGACTGGAAGATGGCAGACCAGTGCGGGCCAGAATGGTCGGACAGGGCAAAGATGAGCACTACGAGCTGTATATCGCCAGAGAGTGGATCGCCCAAGAGTTCGATGCCCTGTGGGCCAGCCAGCAGAGATTTCATGCTGAGGTGCTGGCCGACGCCGCCAGAGATAGACTGAGAGCCATCCTGCTGTTCCAGCGGAAGCTGCTGCCTGTGCCTGTGGGCAAGTGCTTCCTGGAACCTAACCAGCCTAGAGTGGCCGCTGCTCTGCCTAGCGCTCAGAGATTCAGACTGATGCAAGAGCTGAACCACCTGAGAGTGATGACCCTGGCCGACAAGAGAGAGAGGCCCCTGAGCTTCCAAGAGAGAAACGATCTGCTGGCCCAGCTGGTGGCCAGACCTAAGTGCGGCTTCGACATGCTGCGGAAGATCGTGTTCGGCGCCAACAAAGAGGCCTACAGATTCACCATCGAGAGCGAGCGGCGGAAAGAACTGAAGGGCTGTGATACAGCCGCCAAGCTGGCCAAAGTGAATGCCCTGGGAACTAGATGGCAGGCTCTGTCCCTGGACGAGCAGGATAGACTCGTGTGCCTGCTGCTGGACGGCGAGAATGATGCTGTGCTGGCTGATGCCCTGCGGGAACACTATGGACTGACAGACGCCCAGATCGACACACTGCTGGGCCTGTCTTTTGAGGACGGCCACATGAGACTGGGGAGAAGCGCTCTGCTGAGAGTCCTGGATGCCCTGGAATCCGGAAGAGATGAGCAGGGACTGCCCCTGTCCTACGATAAGGCTGTTGTGGCTGCCGGCTATCCAGCTCACACAGCCGATCTGGAAAACGGCGAGAGAGATGCACTGCCCTACTACGGCGAGCTGCTGTGGCGGTATACACAGGATGCCCCTACCGCCAAGAACGACGCCGAGAGAAAGTTCGGCAAGATCGCCAATCCTACCGTGCACATCGGCCTGAATCAGCTGAGAAAGCTTGTCAATGCCCTGATCCAGAGATACGGCAAGCCCGCTCAGATCGTGGTGGAACTGGCCAGAAATCTGAAGGCTGGCCTGGAAGAGAAAGAGCGGATCAAGAAACAGCAGACCGCCAACCTGGAACGGAACGAGAGAATCCGGCAGAAGCTGCAGGACGCTGGCGTGCCCGACAACAGAGAAAACCGGCTGCGGATGCGGCTGTTCGAGGAACTCGGACAAGGCAATGGACTGGGCACCCCTTGCATCTACTCCGGCAGACAGATCAGCCTGCAGAGACTGTTCAGCAACGACGTGCAGGTCGACCACATCCTGCCTTTCAGCAAGACCCTGGATGACAGCTTCGCCAACAAGGTGCTCGCCCAGCACGACGCCAACAGATACAAGGGCAACAGAGGCCCTTTCGAGGCCTTCGGAGCCAACAGAGATGGCTACGCCTGGGACGACATTAGAGCCAGAGCAGCCGTGCTGCCCCGGAACAAGAGAAACAGATTTGCCGAGACAGCCATGCAGGACTGGCTGCACAACGAGACTGACTTTCTGGCTCGGCAGCTGACCGATACCGCCTACCTTAGCAGAGTGGCCAGGCAGTACCTGACCGCCATCTGCAGCAAGGACGACGTGTACGTTAGCCCCGGCAGACTGACTGCCATGCTGAGAGCTAAGTGGGGCCTGAACAGAGTGCTGGATGGCGTGATGGAAGAACAGGGCAGACCCGCCGTGAAGAACCGGGATGATCACAGACACCACGCCATCGACGCCGTGGTTATTGGCGCCACAGATAGAGCCATGCTGCAACAGGTGGCCACACTGGCCGCTAGAGCTAGAGAACAGGACGCCGAAAGGCTGATCGGCGACATGCCTACGCCTTGGCCTAATTTCCTTGAGGACGTGCGGGCTGCCGTGGCCAGATGTGTGGTTTCTCACAAGCCCGACCACGGACCAGAAGGCGGCCTGCATAACGATACAGCCTACGGCATTGTGGCCGGACCATTCGAGGATGGCAGATACAGAGTGCGGCACCGGGTGTCCCTGTTCGATCTGAAACCTGGCGACCTGAGCAACGTCCGCTGTGATGCTCCTCTGCAAGCCGAGCTGGAACCCATCTTCGAGCAGGACGATGCCAGGGCCAGAGAAGTGGCTCTTACAGCCCTGGCTGAGCGGTACAGACAGCGGAAAGTGTGGCTGGAAGAACTGATGAGCGTGCTGCCTATCAGACCCAGAGGCGAGGACGGAAAGACCCTGCCAGATAGCGCTCCTTACAAGGCCTACAAGGGCGACTCCAACTACTGCTATGAGCTGTTCATCAATGAGCGCGGCAGATGGGATGGCGAGCTGATCTCTACCTTCCGGGCCAATCAGGCCGCTTACCGGCGGTTCAGAAATGACCCAGCCAGGTTCAGAAGATACACCGCTGGCGGTAGACCCCTGCTGATGAGACTGTGTATCAACGACTATATCGCCGTGGGCACAGCCGCCGAGAGGACCATCTTTAGAGTGGTCAAGATGAGCGAGAACAAGATCACTCTGGCCGAGCACTTCGAAGGCGGAACCCTGAAACAGAGGGATGCCGACAAGGACGATCCCTTCAAGTATCTGACAAAGAGCCCTGGCGCTCTGCGCGATCTGGGAGCTAGAAGAATCTTCGTGGACCTGATCGGCCGCGTGCTGGACCCAGGCATTAAGGGCGATCCCAAGAAGAAGAGGAAGGTGRsyCas9/NLS protein sequence (SEQ ID NO: 28)MAEKQHRWGLDIGTNSIGWAVIALIEGRPAGLVATGSRIFSDGRNPKDGSSLAVERRGPRQMRRRRDRYLRRRDRPMQALINVGLMPGDAAARKALVTENPYVLRQRGLDQALTLPEFGRALFHLNQRRGFQSNRKTDRATAKESGKVKNAIAAFRAGMGNARTVGEALARRLEDGRPVRARMVGQGKDEHYELYIAREWIAQEFDALWASQQRFHAEVLADAARDRLRAILLFQRKLLPVPVGKCFLEPNQPRVAAALPSAQRFRLMQELNHLRVMTLADKRERPLSFQERNDLLAQLVARPKCGFDMLRKIVFGANKEAYRFTIESERRKELKGCDTAAKLAKVNALGTRWQALSLDEQDRLVCLLLDGENDAVLADALREHYGLTDAQIDTLLGLSFEDGHMRLGRSALLRVLDALESGRDEQGLPLSYDKAVVAAGYPAHTADLENGERDALPYYGELLWRYTQDAPTAKNDAERKFGKIANPTVHIGLNQLRKLVNALIQRYGKPAQIVVELARNLKAGLEEKERIKKQQTANLERNERIRQKLQDAGVPDNRENRLRMRLFEELGQGNGLGTPCIYSGRQISLQRLFSNDVQVDHILPFSKTLDDSFANKVLAQHDANRYKGNRGPFEAFGANRDGYAWDDIRARAAVLPRNKRNRFAETAMQDWLHNETDFLARQLTDTAYLSRVARQYLTAICSKDDVYVSPGRLTAMLRAKWGLNRVLDGVMEEQGRPAVKNRDDHRHHAIDAVVIGATDRAMLQQVATLAARAREQDAERLIGDMPTPWPNFLEDVRAAVARCVVSHKPDHGPEGGLHNDTAYGIVAGPFEDGRYRVRHRVSLFDLKPGDLSNVRCDAPLQAELEPIFEQDDARAREVALTALAERYRQRKVWLEELMSVLPIRPRGEDGKTLPDSAPYKAYKGDSNYCYELFINERGRWDGELISTFRANQAAYRRFRNDPARFRRYTAGGRPLLMRLCINDYIAVGTAAERTIFRVVKMSENKITLAEHFEGGTLKQRDADKDDPFKYLTKSPGALRDLGARRIFVDLIGRVLDPGIKGDPKKKRKVCdiCas9/NLS DNA sequence (SEQ ID NO: 29)ATGAAGTACCACGTGGGCATCGACGTGGGCACCTTTTCTGTTGGACTGGCCGCCATCGAAGTGGACGATGCCGGAATGCCTATCAAGACCCTGAGCCTGGTGTCCCACATCCACGATTCTGGACTGGACCCCGACGAGATCAAGAGCGCCGTTACAAGACTGGCCAGCAGCGGAATCGCCAGAAGAACCAGACGGCTGTACCGGCGGAAGAGAAGAAGGCTGCAGCAGCTGGACAAGTTCATCCAGAGACAAGGCTGGCCCGTGATCGAGCTGGAAGATTACAGCGACCCTCTGTACCCCTGGAAAGTGCGGGCTGAACTGGCTGCCAGCTATATCGCCGATGAGAAAGAGCGGGGCGAGAAGCTGTCTGTGGCCCTGAGACACATTGCCAGACACAGAGGATGGCGGAACCCCTACGCCAAGGTGTCCTCTCTGTATCTGCCTGACGGCCCTAGCGACGCCTTCAAGGCCATCAGAGAGGAAATCAAGAGAGCCAGCGGCCAGCCTGTGCCTGAAACAGCTACAGTGGGCCAGATGGTCACCCTGTGTGAACTGGGCACCCTGAAGTTGAGAGGCGAAGGCGGAGTGCTGTCTGCCAGACTCCAGCAGAGCGATTACGCCAGAGAGATCCAAGAGATTTGCCGGATGCAAGAGATCGGCCAAGAGCTGTACAGAAAGATCATCGATGTGGTGTTCGCCGCCGAGTCTCCTAAGGGATCTGCCTCTAGCAGAGTGGGCAAAGACCCTCTGCAGCCCGGCAAGAATAGAGCCCTGAAAGCCTCCGATGCCTTCCAGAGATACCGGATCGCCGCTCTGATCGGCAACCTGAGAGTTAGAGTGGACGGCGAGAAGAGGATTCTGAGCGTGGAAGAGAAAAACCTGGTGTTCGACCACCTGGTCAATCTGACCCCTAAGAAAGAACCCGAGTGGGTCACAATCGCCGAGATCCTGGGAATCGACAGAGGCCAGCTGATCGGAACCGCCACCATGACAGATGATGGCGAAAGAGCCGGCGCTCGGCCTCCTACACATGACACCAATCGGAGCATCGTGAACAGCAGAATCGCCCCTCTGGTGGACTGGTGGAAAACCGCCTCTGCTCTGGAACAGCACGCTATGGTCAAGGCCCTGTCCAATGCCGAGGTGGACGACTTCGATTCTCCTGAGGGCGCCAAAGTGCAGGCCTTCTTTGCCGACCTGGACGACGATGTGCACGCCAAGCTGGATAGCCTGCATCTGCCTGTTGGCAGAGCCGCCTACAGCGAGGATACACTTGTGCGGCTGACCAGACGGATGCTGAGTGATGGCGTGGACCTGTACACCGCCAGACTGCAAGAGTTTGGCATCGAGCCTAGCTGGACCCCTCCAACACCTAGAATCGGAGAGCCCGTGGGAAACCCCGCTGTGGACAGAGTGCTGAAAACCGTGTCCAGATGGCTGGAAAGCGCCACCAAAACATGGGGCGCTCCCGAGAGAGTGATCATCGAACACGTGCGCGAGGGCTTCGTGACCGAGAAAAGGGCCAGAGAAATGGATGGCGACATGCGGAGAAGGGCCGCCAGAAATGCCAAGCTGTTCCAAGAAATGCAAGAAAAGCTGAACGTGCAGGGCAAGCCCTCCAGAGCCGACCTTTGGAGATACCAGAGCGTGCAGAGACAGAACTGCCAGTGCGCCTACTGTGGCAGCCCTATCACCTTCAGCAACAGCGAGATGGACCACATCGTGCCTAGAGCCGGCCAGGGATCCACCAACACCAGAGAAAATCTGGTGGCCGTGTGCCACAGATGCAACCAGAGCAAGGGCAACACCCCATTCGCCATCTGGGCCAAGAACACCTCTATCGAGGGCGTGTCCGTGAAAGAAGCCGTGGAAAGAACCAGGCACTGGGTCACCGATACCGGCATGAGAAGCACCGACTTCAAGAAATTCACCAAGGCCGTGGTGGAACGGTTCCAGAGGGCCACAATGGACGAGGAAATTGACGCCCGCAGCATGGAAAGCGTGGCCTGGATGGCCAATGAGCTGAGAAGTAGAGTGGCCCAGCACTTCGCCAGCCACGGCACAACAGTCAGAGTGTACAGAGGCAGCCTGACCGCCGAAGCTCGTAGAGCCTCTGGAATCAGCGGCAAGCTGAAGTTCTTTGACGGCGTGGGCAAGAGCAGACTGGACAGAAGGCACCACGCCATTGATGCCGCCGTGATCGCCTTCACCAGCGACTATGTGGCCGAAACACTGGCCGTGCGGAGCAACCTCAAACAGAGCCAGGCTCACAGACAAGAGGCTCCTCAGTGGCGCGAGTTCACAGGCAAAGATGCCGAACACAGAGCCGCTTGGAGAGTGTGGTGCCAGAAGATGGAAAAACTGAGCGCCCTGCTGACCGAGGACCTGAGAGATGATAGAGTGGTGGTCATGAGCAACGTGCGCCTGAGACTCGGAAATGGCAGCGCCCACAAAGAGACAATCGGAAAGCTGAGCAAAGTGAAGCTGTCCAGCCAGCTGAGCGTGTCCGACATCGATAAGGCCAGCTCTGAGGCCCTTTGGTGCGCCCTGACAAGAGAACCTGGCTTCGACCCCAAAGAGGGACTGCCTGCCAATCCTGAGCGGCACATCAGAGTGAATGGCACCCATGTGTACGCCGGCGACAACATCGGCCTGTTTCCAGTGTCTGCCGGATCTATCGCTCTGAGAGGCGGATATGCCGAGCTGGGCAGCTCTTTCCATCACGCCAGGGTGTACAAGATCACAAGCGGCAAGAAACCCGCCTTTGCCATGCTGAGAGTGTATACCATCGACCTGCTGCCTTACCGGAACCAGGACCTGTTCAGCGTGGAACTGAAGCCCCAGACCATGAGCATGAGACAGGCCGAGAAGAAGCTGAGGGACGCCCTGGCTACAGGCAACGCCGAATATCTTGGATGGCTGGTGGTGGATGACGAGCTGGTGGTCGATACCAGCAAGATCGCCACCGACCAAGTGAAGGCTGTGGAAGCCGAACTGGGAACCATCAGACGTTGGCGCGTGGACGGCTTTTTCAGCCCCTCTAAGCTGAGACTGCGGCCCCTGCAGATGAGCAAAGAGGGCATCAAGAAAGAGAGCGCCCCTGAGCTGTCCAAGATCATTGACAGACCTGGCTGGCTGCCCGCCGTGAACAAGCTGTTTTCTGACGGCAACGTGACCGTCGTGCGGAGAGATTCTCTGGGCAGAGTGCGCCTGGAAAGCACAGCACATCTGCCCGTGACATGGAAGGTGCAGCCCAAGAAGAAGAGGAAGGTGCdiCas9/NLS protein sequence (SEQ ID NO: 30)MKYHVGIDVGTFSVGLAAIEVDDAGMPIKTLSLVSHIHDSGLDPDKIKSAVTRLASSGIARRTRRLYRRKRRRLQQLDKFIQRQGWPVIELEDYSDPLYPWKVRAELAASYIADEKERGEKLSVALRHIARHRGWRNPYAKVSSLYLPDEPSDAFKAIREEIKRASGQPVPETATVGQMVTLCELGTLKLRGEGGVLSARLQQSDHAREIQEICRMQEIGQELYRKIIDVVFAAESPKGSASSRVGKDPLQPGKNRALKASDAFQRYRIAALIGNLRVRVDGEKRILSVEEKNLVFDHLVNLAPKKEPEWVTIAEILGIDRGQLIGTATMTDDGERAGARPPTHDTNRSIVNSRIAPLVDWWKTASALEQHAMVKALSNAEVDDFDSPEGAKVQAFFADLDDDVHAKLDSLHLPVGRAAYSEDTLVRLTRRMLADGVDLYTARLQEFGIEPSWTPPAPRIGEPVGNPAVDRVLKTVSRWLESATKTWGAPERVIIEHVREGFVTEKRAREMDGDMRRRAARNAKLFQEMQEKLNVQGKPSRADLWRYQSVQRQNCQCAYCGSPITFSNSEMDHIVPRAGQGSTNTRENLVAVCHRCNQSKGNTPFAIWAKNTSIEGVSVKEAVERTRHWVTDTGMRSTDFKKFTKAVVERFQRATMDEEIDARSMESVAWMANELRSRVAQHFASHGTTVRVYRGSLTAEARRASGISGKLEFLDGVGKSRLDRRHHAIDAAVIAFTSDYVAETLAVRSNLKQSQAHRQEAPQWREFTGKDAEHRAAWRVWCQKMEKLSALLTEDLRDDRVVVMSNVRLRLGNGSAHEETIGKLSKVKLGSQLSVSDIDKASSEALWCALTREPDFDPKDGLPANPERHIRVNGTHVYAGDNIGLFPVSAGSIALRGGYAELGSSFHHARVYKITSGKKPAFAMLRVYTIDLLPYRNQDLFSVELKPQTMSMRQAEKKLRDALATGNAEYLGWLVVDDELVVDTSKIATDQVKAVEAELGTIRRWRVDGFFGDTRLRLRPLQMSKEGIKKESAPELSKIIDRPGWLPAVNKLFSEGNVTVVRRDSLGRVRLESTAHLPVTWKVQPKKKRKV Bsm Cas9 sgRNA (SEQ ID NO: No 31)NNNNNNNNNNNNNNNNNNNNGUCAUAGUUCCCCUAAGAUUAUUGAAACAAUGAUCUUAGGGUUACUAUGAUAAGGGCUUUCUACUUUAGGGGUAGAGAUGUCCCGCGGCGUUGGGGAUCGCCUAUUGCCCUUAAAGGGCACUCCCCAUUUUAAUUUUUUU Lrh Cas9 sgRNA (SEQ ID NO: 32)NNNNNNNNNNNNNNNNNNNNGUCUCAGGUAGAUGUCAGAUCAAUCAGAAAUGAUUGAUCUGACAUCUACGAGUUGAGAUCAAACAAAGCUUCAGCUGAGUUUCAAUUUCUGAGCCCAUGUUGGGCCAUACAUAUGCCACCCGAGUGCAAAUCGGGUGGCUUUUUUU Pex Cas9 sgRNA (SEQ ID NO: 33)NNNNNNNNNNNNNNNNNNNNGUUUCAGUAGUUGUUAGAAGAAUGAAAAUUCUUUUAACAACGAAGUCGCCUUCGGGCGAGCUGAAAUCAAUUUGAUUAAAUAUUAGAUCCGGCUACUGAGGUCUUUGACCUUAUCCGGAUUAACGAAGAGCCUCCGAGGAGGCUUUUUUUMca Cas9 sgRNA (SEQ ID NO: 34)NNNNNNNNNNNNNNNNNNNNGUUUUAGUGUUGUACAAUAUUUGGGUGAAAACCCAAAUAUUGUACAUCCUAAAUCAAGGCGCUUAAUUGCUGCCGUAAUUGCUGAAAGCGUAGCUUUCAGUUUUUU UMga Cas9 sgRNA (SEQ ID NO: 35)NNNNNNNNNNNNNNNNNNNNGUUUUAGCACUGUACAAUACUUGUGUAAGCAAUAACGAAAAUUAUUGCUUACACAAUUAUUGUCGUGCUAAAAUAAGGCGCUGUUAAUGCAGCUGCCGCAUCCGCCAGAGCAUUUAUGCUCUGGCUUUUUUU Agl Cas9 sgRNA (SEQ ID NO: 36)NNNNNNNNNNNNNNNNNNNNGUUUUGCCUUGAAUCCAAAGUAAGGCAUGGUAgaaaUAUUAUUCCUGUGGAUUCAAGACAAAAUUUGAAAUGCAAACCGAUUCCCCGGCUGCAAGCCAGCCACACCGGUCUUUCAAAGCAUUUUUUU Amu Cas9 sgRNA (SEQ ID NO: 37)NNNNNNNNNNNNNNNNNNNNGUUUUGCCUUGAAUCCAAAACGGAUUCAAGACAAAAUUUGAAAUGCAAACCGAUUUUCCUGACUGCCAGCCAGUCACACCGGUAACAAAAGCAUUUUUUUOki Cas9 sgRNA (SEQ ID NO: 38)NNNNNNNNNNNNNNNNNNNNGCUUCAGAUGUGUGUCAGAUCAAUGAgaaaUCAUUGAUCUGACACACAGCAUUGAAGUAAAGCAAGAUUAAUUUCAAGCUUAAUUUUCUUCACAUUUUAUGUGCAGAAGGGCUUAUGCCCACAAUACAUAAAAAGUCCGCAUUCACUUGCGGACUUUUAUUUUUUUBbo Cas9 sgRNA (SEQ ID NO: 39)NNNNNNNNNNNNNNNNNNNNGUUUCAAAUUCAAUCUAAAGCGAAAGCUAUACUUAUUAUUGAAUUUGAAAUAAGGCUGUUCCUUCGUUAGUUCAGUCGAUUGCUCCUCCGGUAUUGCUUAUGCAUGCCGGAGUUUUUU Ace Cas9 sgRNA (SEQ ID NO: 40)NNNNNNNNNNNNNNNNNNNNGCUGGGGAGCCUGUCUGAAAAGACAGGCUACCUAGCAAGACCCCUUCGUGGGGUCGCAUUCUUCACCCCCUCGCAGCAGCGAGGGGGUUCGUUUUUUUAhe Cas9 sgRNA (SEQ ID NO: 41)NNNNNNNNNNNNNNNNNNNNGUCAUAGUUCCCUCACAAGCCUCGAUGUGGAAACACAUCAAGGCUUGCGAGGUUGCUAUGAUAAGGCAACAGGCCGCAAAGCACUGACCCGCAUUCCAAUGAAUGCGGGUCAUCUACUUUUUUU Wsu Cas9 sgRNA (SEQ ID NO: 42)NNNNNNNNNNNNNNNNNNNNGUUUCACAGGCUAAGCGGAUUUGCgaaaGCAAAUCCGUUCGAUGCCUUGAAAUCAUCAAAAAGAUAUAAUAGACCCGCCCACUGUAUUGUACAUGGCGGGACUUUU UUUNsa Cas9 sgRNA (SEQ ID NO: 43)NNNNNNNNNNNNNNNNNNNNGUUAUAAGACCCCUCAAAACCCCACCCUGUUACAAUGUUGUAACAGGGUAGGGUUAUUUGAGGGGUCUUAUAAUCAAGAACUGUUACAACAGUUCCAUUCUAGGGCCCAUCUUCGGACGGGCCUCAGCCUUUUUUU Rsy Cas9 sgRNA (SEQ ID NO: 44)NNNNNNNNNNNNNNNNNNNNGUUGUAGCCAGAGCGCAAUUCCCGAUCUGCUGAAAAGCAGAUCGGGAAUUGCGCUUUGCUACUAACAAGCUGAAUCCGUUAGGAGUAAAUGCACCAAAUGAGAGGGCCGGCUUAUGCCGGCCCUUUGCUUUUUUU Cdi Cas9 sgRNA (SEQ ID NO: 45)NNNNNNNNNNNNNNNNNNNNACUGGGGUUCAGUUCUCAAAAACCCUGAUAGACUUCGAAAAGUCACUAACUUAAUUAAAUAGAACUGAACCUCAGUAAGCAUUGGCUCGUUUCCAAUGUUGAUUGCUCCGCCGGUGCUCCUUAUUAUUAAGGGCGCCGGCUUUCUUUUUUUPexCas9-HN1HB1 fusion (SEQ ID NO: 117)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSD TGSGMGKTHIIGVGLDLGGTYTGTFITSHPSDEAEHRDHSSAFTVVNSEKLSFSSKSRTAVRHRVRSYKGFDLRRRLLLLVAEYQLLQKKQTLAPEERENLRIALSGYLKRRGYARTEAETDTSVLESLDPSVFSSAPSFTNFFNDSEPLNIQWEAIANSPETTKALNKELSGQKEADFKKYIKTSFPEYSAKEILANYVEGRRAILDASKYIANLQSLGHKHRSKYLSDILQDMKRDSRITRLSEAFGSTDNLWRIIGNISNLQERAVRWYFNDAKFEQGQEQLDAVKLKNVLVRALKYLRSDDKEWSASQKQIIQSLEQSGDVLDVLAGLDPDRTIPPYEDQNNRRPPEDQTLYLNPKALSSEYGEKWKSWANKFAGAYPLLTEDLTEILKNTDRKSRIKIRSDVLPDSDYRLAYILQRAFDRSIALDECSIRRTAEDFENGVVIKNEKLEDVLSGHQLEEFLEFANRYYQETAKAKNGLWFPENALLERADLHPPMKNKILNVIVGQALGVSPAEGTDFIEEIWNSKVKGRSTVRSICNAIENERKTYGPYFSEDYKFVKTALKEGKTEKELSKKFAAVIKVLKMVSEVVPFIGKELRLSDEAQSKFDNLYSLAQLYNLIETERNGFSKVSLAAHLENAWRMTMTDGSAQCCRLPADCVRPFDGFIRKAIDRNSWEVAKRIAEEVKKSVDFTNGTVKIPVAIEANSFNFTASLTDLKYIQLKEQKLKKKLEDIQRNEENQEKRWLSKEERIRADSHGICAYTGRPLDDVGEIDHIIPRSLTLKKSESIYNSEVNLIFVSAQGNQEKKNNIYLLSNLAKNYLAAVFGTSDLSQITNEIESTVLQLKAAGRLGYFDLLSEKERACARHALFLNSDSEARRAVIDVLGSRRKASVNGTQAWFVRSIFSKVRQALAAWTQETGNELIFDAISVPAADSSEMRKRFAEYRPEFRKPKVQPVASHSIDAMCIYLAACSDPFKTKRMGSQLAIYEPINFDNLFTGSCQVIQNTPRNFSDKTNIANSPIFKETIYAERFLDIIVSRGEIFIGYPSNMPFEEKPNRISIGGKDPFSILSVLGAYLDKAPSSEKEKLTIYRVVKNKAFELFSKVAGSKFTAEEDKAAKILEALHFVTVKQDVAATVSDLIKSKKELSKDSIENLAKQKGCLKKVEYSSKEFKFKGSLIIPAAVEWGKVLWNVFKENTAEELKDENALRKALEAAWPSSFGTRNLHSKAKRVFSLPVVATQSGAVRIRRKTAFGDFVYQSQDTNNLYSSFPVKNGKLDWSSPIIHPALQNRNLTAYGYRFVDHDRSISMSEFREVYNKDDLMRIELAQGTSSRRYLRVEMPGEKFLAWFGENSISLGSSFKFSVSEVFDNKIYTENAEFTKFLPKPREDNKHNGTIFFELVGPRVIFNYIVGGAASSLKEIFSEAGKERSPKKKRKV LEGGGGS GKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGEPexCas9-HN1H1G fusion (SEQ ID NO: 118)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSD TGSGMGKTHIIGVGLDLGGTYTGTFITSHPSDEAEHRDHSSAFTVVNSEKLSFSSKSRTAVRHRVRSYKGFDLRRRLLLLVAEYQLLQKKQTLAPEERENLRIALSGYLKRRGYARTEAETDTSVLESLDPSVFSSAPSFTNFFNDSEPLNIQWEAIANSPETTKALNKELSGQKEADFKKYIKTSFPEYSAKEILANYVEGRRAILDASKYIANLQSLGHKHRSKYLSDILQDMKRDSRITRLSEAFGSTDNLWRIIGNISNLQERAVRWYFNDAKFEQGQEQLDAVKLKNVLVRALKYLRSDDKEWSASQKQIIQSLEQSGDVLDVLAGLDPDRTIPPYEDQNNRRPPEDQTLYLNPKALSSEYGEKWKSWANKFAGAYPLLTEDLTEILKNTDRKSRIKIRSDVLPDSDYRLAYILQRAFDRSIALDECSIRRTAEDFENGVVIKNEKLEDVLSGHQLEEFLEFANRYYQETAKAKNGLWFPENALLERADLHPPMKNKILNVIVGQALGVSPAEGTDFIEEIWNSKVKGRSTVRSICNAIENERKTYGPYFSEDYKFVKTALKEGKTEKELSKKFAAVIKVLKMVSEVVPFIGKELRLSDEAQSKFDNLYSLAQLYNLIETERNGFSKVSLAAHLENAWRMTMTDGSAQCCRLPADCVRPFDGFIRKAIDRNSWEVAKRIAEEVKKSVDFTNGTVKIPVAIEANSFNFTASLTDLKYIQLKEQKLKKKLEDIQRNEENQEKRWLSKEERIRADSHGICAYTGRPLDDVGEIDHIIPRSLTLKKSESIYNSEVNLIFVSAQGNQEKKNNIYLLSNLAKNYLAAVFGTSDLSQITNEIESTVLQLKAAGRLGYFDLLSEKERACARHALFLNSDSEARRAVIDVLGSRRKASVNGTQAWFVRSIFSKVRQALAAWTQETGNELIFDAISVPAADSSEMRKRFAEYRPEFRKPKVQPVASHSIDAMCIYLAACSDPFKTKRMGSQLAIYEPINFDNLFTGSCQVIQNTPRNFSDKTNIANSPIFKETIYAERFLDIIVSRGEIFIGYPSNMPFEEKPNRISIGGKDPFSILSVLGAYLDKAPSSEKEKLTIYRVVKNKAFELFSKVAGSKFTAEEDKAAKILEALHFVTVKQDVAATVSDLIKSKKELSKDSIENLAKQKGCLKKVEYSSKEFKFKGSLIIPAAVEWGKVLWNVFKENTAEELKDENALRKALEAAWPSSFGTRNLHSKAKRVFSLPVVATQSGAVRIRRKTAFGDFVYQSQDTNNLYSSFPVKNGKLDWSSPIIHPALQNRNLTAYGYRFVDHDRSISMSEFREVYNKDDLMRIELAQGTSSRRYLRVEMPGEKFLAWFGENSISLGSSFKFSVSEVFDNKIYTENAEFTKFLPKPREDNKHNGTIFFELVGPRVIFNYIVGGAASSLKEIFSEAGKERSPKKKRKV LEGGGGS STDHPKYSDMIVAAIQAEKNRAGSSRQSIQKYIKSHYKVGENADSQIKLSIKRLVTTGVLKQTKGVGASGSFRLAKSDEPBsmCas9-HN1HB1 fusion (SEQ ID NO: 119)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSD TGSGMNYKMGLDIGIASVGWAVINLDLKRIEDLGVRIFDKAEHPQNGESLALPRRIARSARRRLRRRKHRLERIRRLLVSENVLTKEEMNLLFKQKKQIDVWQLRVDALERKLNNDELARVLLHLAKRRGFKSNRKSERNSKESSEFLKNIEENQSILAQYRSVGEMIVKDSKFAYHKRNKLDSYSNMIARDDLEREIKLIFEKQREFNNPVCTERLEEKYLNIWSSQRPFASKEDIEKKVGFCTFEPKEKRAPKATYTFQSFIVWEHINKLRLVSPDETRALTEIERNLLYKQAFSKNKMTYYDIRKLLNLSDDIHFKGLLYDPKSSLKQIENIRFLELDSYHKIRKCIENVYGKDGIRMFNETDIDTFGYALTIFKDDEDIVAYLQNEYITKNGKRVSNLANKVYDKSLIDELLNLSFSKFAHLSMKAIRNILPYMEQGEIYSKACELAGYNFTGPKKKEKALLLPVIPNIANPVVMRALTQSRKVVNAIIKKYGSPVSIHIELARDLSHSFDERKKIQKDQTENRKKNETAIKQLIEYELTKNPTGLDIVKFKLWSEQQGRCMYSLKPIELERLLEPGYVEVDHILPYSRSLDDSYANKVLVLTKENREKGNHTPVEYLGLGSERWKKFEKFVLANKQFSKKKKQNLLRLRYEETEEKEFKERNLNDTRYISKFFANFIKEHLKFADGDGGQKVYTINGKITAHLRSRWDFNKNREESDLHHAVDAVIVACATQGMIKKITEFYKAREQNKESAKKKEPIFPQPWPHFADELKARLSKFPQESIEAFALGNYDRKKLESLRFVFVSRMPKRSVTGAAHQETLRRCVGIDEQSGKIQTAVKTKLSDIKLDKDGHFPMYQKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNKVVHLDGSKTVAYNSNIVRTDVFEKDGKYYCVPVYTMDIMKGTLPNKAIEANKPYSEWKEMTEEYTFQFSLFPNDLVRIVLPREKTIKTSTNEEIIIKDIFAYYKTIDSATGGLELISHDRNFSLRGVGSKTLKRFEKYQVDVLGNIHKVKGEKRVGLAAPTNQKKGKTVDSLQSVSDPKKKR KVLEGGGGS GKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGE BsmCas9-HN1H1G fusion (SEQ ID NO: 120)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSDTGSGMNYKMGLDIGIASVGWAVINLDLKRIEDLGVRIFDKAEHPQNGESLALPRRIARSARRRLRRRKHRLERIRRLLVSENVLTKEEMNLLFKQKKQIDVWQLRVDALERKLNNDELARVLLHLAKRRGFKSNRKSERNSKESSEFLKNIEENQSILAQYRSVGEMIVKDSKFAYHKRNKLDSYSNMIARDDLEREIKLIFEKQREFNNPVCTERLEEKYLNIWSSQRPFASKEDIEKKVGFCTFEPKEKRAPKATYTFQSFIVWEHINKLRLVSPDETRALTEIERNLLYKQAFSKNKMTYYDIRKLLNLSDDIHFKGLLYDPKSSLKQIENIRFLELDSYHKIRKCIENVYGKDGIRMFNETDIDTFGYALTIFKDDEDIVAYLQNEYITKNGKRVSNLANKVYDKSLIDELLNLSFSKFAHLSMKAIRNILPYMEQGEIYSKACELAGYNFTGPKKKEKALLLPVIPNIANPVVMRALTQSRKVVNAIIKKYGSPVSIHIELARDLSHSFDERKKIQKDQTENRKKNETAIKQLIEYELTKNPTGLDIVKFKLWSEQQGRCMYSLKPIELERLLEPGYVEVDHILPYSRSLDDSYANKVLVLTKENREKGNHTPVEYLGLGSERWKKFEKFVLANKQFSKKKKQNLLRLRYEETEEKEFKERNLNDTRYISKFFANFIKEHLKFADGDGGQKVYTINGKITAHLRSRWDFNKNREESDLHHAVDAVIVACATQGMIKKITEFYKAREQNKESAKKKEPIFPQPWPHFADELKARLSKFPQESIEAFALGNYDRKKLESLRFVFVSRMPKRSVTGAAHQETLRRCVGIDEQSGKIQTAVKTKLSDIKLDKDGHFPMYQKESDPRTYEAIRQRLLEHNNDPKKAFQEPLYKPKKNGEPGPVIRTVKIIDTKNKVVHLDGSKTVAYNSNIVRTDVFEKDGKYYCVPVYTMDIMKGTLPNKAIEANKPYSEWKEMTEEYTFQFSLFPNDLVRIVLPREKTIKTSTNEEIIIKDIFAYYKTIDSATGGLELISHDRNFSLRGVGSKTLKRFEKYQVDVLGNIHKVKGEKRVGLAAPTNQKKGKTVDSLQSVSDPKKKR KVLEGGGGS STDHPKYSDMIVAAIQAEKNRAGSSRQSIQKYIKSHYKVGENADSQIKLSIKRLVTTGVLKQTKGVGASGSFRLAKSDEP LrhCas9-HN1HB1 fusion (SEQ ID NO: 121)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSD TGSGMTKLNQPYGIGLDIGSNSIGFAVVDANSHLLRLKGETAIGARLFREGQSAADRRGSRTTRRRLSRTRWRLSFLRDFFAPHITKIDPDFFLRQKYSEISPKDKDRFKYEKRLFNDRTDAEFYEDYPSMYHLRLHLMTHTHKADPREIFLAIHHILKSRGHFLTPGAAKDFNTDKVDLEDIFPALTEAYAQVYPDLELTFDLAKADDFKAKLLDEQATPSDTQKALVNLLLSSDGEKEIVKKRKQVLTEFAKAITGLKTKFNLALGTEVDEADASNWQFSMGQLDDKWSNIETSMTDQGTEIFEQIQELYRARLLNGIVPAGMSLSQAKVADYGQHKEDLELFKTYLKKLNDHELAKTIRGLYDRYINGDDAKPFLREDFVKALTKEVTAHPNEVSEQLLNRMGQANFMLKQRTKANGAIPIQLQQRELDQIIANQSKYYDWLAAPNPVEAHRWKMPYQLDELLNFHIPYYVGPLITPKQQAESGENVFAWMVRKDPSGNITPYNFDEKVDREASANTFIQRMKTTDTYLIGEDVLPKQSLLYQKYEVLNELNNVRINNECLGTDQKQRLIREVFERHSSVTIKQVADNLVAHGDFARRPEIRGLADEKRFLSSLSTYHQLKEILHEAIDDPTKLLDIENIITWSTVFEDHTIFETKLAEIEWLDPKKINELSGIRYRGWGQFSRKLLDGLKLGNGHTVIQELMLSNHNLMQILADETLKETMTELNQDKLKTDDIEDVINDAYTSPSNKKALRQVLRVVEDIKHAANGQDPSWLFIETADGTGTAGKRTQSRQKQIQTVYANAAQELIDSAVRGELEDKIADKASFTDRLVLYFMQGGRDIYTGAPLNIDQLSHYDIDHILPQSLIKDDSLDNRVLVNATINREKNNVFASTLFAGKMKATWRKWHEAGLISGRKLRNLMLRPDEIDKFAKGFVARQLVETRQIIKLTEQIAAAQYPNTKIIAVKAGLSHQLREELDFPKNRDVNHYHHAFDAFLAARIGTYLLKRYPKLAPFFTYGEFAKVDVKKFREFNFIGALTHAKKNIIAKDTGEIVWDKERDIRELDRIYNFKRMLITHEVYFETADLFKQTIYAAKDSKERGGSKQLIPKKQGYPTQVYGGYTQESGSYNALVRVAEADTTAYQVIKISAQNASKIASANLKSREKGKQLLNEIVVKQLAKRRKNWKPSANSFKIVIPRFGMGTLFQNAKYGLFMVNSDTYYRNYQELWLSRENQKLLKKLFSIKYEKTQMNHDALQVYKAIIDQVEKFFKLYDINQFRAKLSDAIERFEKLPINTDGNKIGKTETLRQILIGLQANGTRSNVKNLGIKTDLGLLQVGSGIKLDKDTQIVYQSPSGLFKRRIPLADLPKKKRKV LEGGGGS GKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGELrhCas9-HN1H1G fusion (SEQ ID NO: 122)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSD TGSGMTKLNQPYGIGLDIGSNSIGFAVVDANSHLLRLKGETAIGARLFREGQSAADRRGSRTTRRRLSRTRWRLSFLRDFFAPHITKIDPDFFLRQKYSEISPKDKDRFKYEKRLFNDRTDAEFYEDYPSMYHLRLHLMTHTHKADPREIFLAIHHILKSRGHFLTPGAAKDFNTDKVDLEDIFPALTEAYAQVYPDLELTFDLAKADDFKAKLLDEQATPSDTQKALVNLLLSSDGEKEIVKKRKQVLTEFAKAITGLKTKFNLALGTEVDEADASNWQFSMGQLDDKWSNIETSMTDQGTEIFEQIQELYRARLLNGIVPAGMSLSQAKVADYGQHKEDLELFKTYLKKLNDHELAKTIRGLYDRYINGDDAKPFLREDFVKALTKEVTAHPNEVSEQLLNRMGQANFMLKQRTKANGAIPIQLQQRELDQIIANQSKYYDWLAAPNPVEAHRWKMPYQLDELLNFHIPYYVGPLITPKQQAESGENVFAWMVRKDPSGNITPYNFDEKVDREASANTFIQRMKTTDTYLIGEDVLPKQSLLYQKYEVLNELNNVRINNECLGTDQKQRLIREVFERHSSVTIKQVADNLVAHGDFARRPEIRGLADEKRFLSSLSTYHQLKEILHEAIDDPTKLLDIENIITWSTVFEDHTIFETKLAEIEWLDPKKINELSGIRYRGWGQFSRKLLDGLKLGNGHTVIQELMLSNHNLMQILADETLKETMTELNQDKLKTDDIEDVINDAYTSPSNKKALRQVLRVVEDIKHAANGQDPSWLFIETADGTGTAGKRTQSRQKQIQTVYANAAQELIDSAVRGELEDKIADKASFTDRLVLYFMQGGRDIYTGAPLNIDQLSHYDIDHILPQSLIKDDSLDNRVLVNATINREKNNVFASTLFAGKMKATWRKWHEAGLISGRKLRNLMLRPDEIDKFAKGFVARQLVETRQIIKLTEQIAAAQYPNTKIIAVKAGLSHQLREELDFPKNRDVNHYHHAFDAFLAARIGTYLLKRYPKLAPFFTYGEFAKVDVKKFREFNFIGALTHAKKNIIAKDTGEIVWDKERDIRELDRIYNFKRMLITHEVYFETADLFKQTIYAAKDSKERGGSKQLIPKKQGYPTQVYGGYTQESGSYNALVRVAEADTTAYQVIKISAQNASKIASANLKSREKGKQLLNEIVVKQLAKRRKNWKPSANSFKIVIPRFGMGTLFQNAKYGLFMVNSDTYYRNYQELWLSRENQKLLKKLFSIKYEKTQMNHDALQVYKAIIDQVEKFFKLYDINQFRAKLSDAIERFEKLPINTDGNKIGKTETLRQILIGLQANGTRSNVKNLGIKTDLGLLQVGSGIKLDKDTQIVYQSPSGLFKRRIPLADLPKKKRKV LEGGGGS STDHPKYSDMIVAAIQAEKNRAGSSRQSIQKYIKSHYKVGENADSQIKLSIKRLVTTGVLKQTKGVGASGSFRLAKSDEPMcaCas9-HN1HB1 fusion (SEQ ID NO: 123)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSD TGSGMEKKRKVTLGFDLGIASVGWAIVDSETNQVYKLGSRLFDAPDTNLERRTQRGTRRLLRRRKYRNQKFYNLVKRTEVFGLSSREAIENRFRELSIKYPNIIELKTKALSQEVCPDEIAWILHDYLKNRGYFYDEKETKEDFDQQTVESMPSYKLNEFYKKYGYFKGALSQPTESEMKDNKDLKEAFFFDFSNKEWLKEINYFFNVQKNILSETFIEEFKKIFSFTRDISKGPGSDNMPSPYGIFGEFGDNGQGGRYEHIWDKNIGKCSIFTNEQRAPKYLPSALIFNFLNELANIRLYSTDKKNIQPLWKLSSIDKLNILLNLFNLPISEKKKKLTSTNINDIVKKESIKSIMLSVEDIDMIKDEWAGKEPNVYGVGLSGLNIEESAKENKFKFQDLKILNVLINLLDNVGIKFEFKDRSDIIKNLELLDNLYLFLIYQKESNNKDSSIDLFIAKNKSLNIENLKLKLKEFLLGAGNEFENHNSKTHSLSKKAIDAILPKLLDNNEGWNLEAIKNYDEEIKSQIEDNSSLMAKQDKKYLNDNFLKDAILPPNVKVTFQQAILIFNKIIQKFSKDFEIDKVVIELAREMTQDQENDALKGIAKAQKSKKSLVEERLEANNIDKSVFNDKYEKLIYKIFLWISQDFKDPYTGAKISANEIVDNKVEIDHIIPYSLCFDDSSANKVLVHKQSNQEKSNSLPYEYIKQGHSGWNWDEFTKYVKRVFVNNVDSILSKKERLKKSENLLTTSYDGYEKLGFLARNLNDTRYATILFRDQLNNYAEHHLIDNKKMFKVIAMNGAVTSFIRKNMSYDNKLRLKDRSDFSHHAYDAAIIALFSNKTKTLYNLIDPSLNGIISKRSEGYWVIEDRYTGEIKELKKEDWTSIKNNVQARKIAKEIEEYLIDLDDEVFFSRKTKRKTNRQLYNETIYGIAAKTDEDGITNYYKKEKFSILDDKDIYLRLLREREKFVINQSNPEVIDQIIEIIESYGKENNIPSRDEAINIKYTKNKINYNLYLKQYMRSLTKSLDQFSEGFINQMIANKTFVLYNPTKNTTRKIKFLRLVNDVKINDIRKNQVINKFNGKNNEPKAFYENINSLGAIVFKSSANNFKTLSINTQIAIFGDKNWDIEDFKTYNMEKIEKYKEIYGIDKTYNFHSFIFPGTILLDKQNKEFYYISSIQTVNDQIELKFLNKIEFKNDDNTSGANKPPRRLRFGIKSIMNNYEQVDISPFGINKKIFEPKKKRKV LEGGGGS GKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCSERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGEMcaCas9-HN1H1G fusion (SEQ ID NO: 124)MPKRKVSSAEGAAKEEPKRRSARLSAKPPAKVEAKPKKAAAKDKSSDKKVQTKGKRGAKGKQAEVANQETKEDLPAENGETKTEESPASDEAGEKEAKSD TGSGMEKKRKVTLGFDLGIASVGWAIVDSETNQVYKLGSRLFDAPDTNLERRTQRGTRRLLRRRKYRNQKFYNLVKRTEVFGLSSREAIENRFRELSIKYPNIIELKTKALSQEVCPDEIAWILHDYLKNRGYFYDEKETKEDFDQQTVESMPSYKLNEFYKKYGYFKGALSQPTESEMKDNKDLKEAFFFDFSNKEWLKEINYFFNVQKNILSETFIEEFKKIFSFTRDISKGPGSDNMPSPYGIFGEFGDNGQGGRYEHIWDKNIGKCSIFTNEQRAPKYLPSALIFNFLNELANIRLYSTDKKNIQPLWKLSSIDKLNILLNLFNLPISEKKKKLTSTNINDIVKKESIKSIMLSVEDIDMIKDEWAGKEPNVYGVGLSGLNIEESAKENKFKFQDLKILNVLINLLDNVGIKFEFKDRSDIIKNLELLDNLYLFLIYQKESNNKDSSIDLFIAKNKSLNIENLKLKLKEFLLGAGNEFENHNSKTHSLSKKAIDAILPKLLDNNEGWNLEAIKNYDEEIKSQIEDNSSLMAKQDKKYLNDNFLKDAILPPNVKVTFQQAILIFNKIIQKFSKDFEIDKVVIELAREMTQDQENDALKGIAKAQKSKKSLVEERLEANNIDKSVFNDKYEKLIYKIFLWISQDFKDPYTGAKISANEIVDNKVEIDHIIPYSLCFDDSSANKVLVHKQSNQEKSNSLPYEYIKQGHSGWNWDEFTKYVKRVFVNNVDSILSKKERLKKSENLLTTSYDGYEKLGFLARNLNDTRYATILFRDQLNNYAEHHLIDNKKMFKVIAMNGAVTSFIRKNMSYDNKLRLKDRSDFSHHAYDAAIIALFSNKTKTLYNLIDPSLNGIISKRSEGYWVIEDRYTGEIKELKKEDWTSIKNNVQARKIAKEIEEYLIDLDDEVFFSRKTKRKTNRQLYNETIYGIAAKTDEDGITNYYKKEKFSILDDKDIYLRLLREREKFVINQSNPEVIDQIIEIIESYGKENNIPSRDEAINIKYTKNKINYNLYLKQYMRSLTKSLDQFSEGFINQMIANKTFVLYNPTKNTTRKIKFLRLVNDVKINDIRKNQVINKFNGKNNEPKAFYENINSLGAIVFKSSANNFKTLSINTQIAIFGDKNWDIEDFKTYNMEKIEKYKEIYGIDKTYNFHSFIFPGTILLDKQNKEFYYISSIQTVNDQIELKFLNKIEFKNDDNTSGANKPPRRLRFGIKSIMNNYEQVDISPFGINKKIFEPKKKRKV LEGGGGS STDHPKYSDMIVAAIQAEKNRAGSSRQSIQKYIKSHYKVGENADSQIKLSIKRLVTTGVLKQTKGVGASGSFRLAKSDEP

What is claimed is:
 1. A system for eukaryotic genome modificationcomprising (a) an engineered Bacillus smithii Cas9 protein comprising anuclear localization signal (NLS), and (b) an engineered guide RNA,wherein the engineered guide RNA is designed to complex with theengineered Bacillus smithii Cas9 protein and the engineered guide RNAcomprises a 5′ guide sequence designed to hybridize with a targetsequence in a double-stranded sequence, and wherein the target sequenceis 5′ to a protospacer adjacent motif (PAM) comprising the sequence5′-NNNNCAAA-3′, wherein N is A, C, G, or T.
 2. The system of claim 1,wherein the engineered Bacillus smithii Cas9 protein further comprisesat least one heterologous domain and the at least one heterologousdomain is a cell-penetrating domain, a marker domain, a chromatinmodulating motif, an RNA aptamer binding domain, or combination thereof.3. The system of claim 1, wherein the engineered Bacillus smithii Cas9protein further comprises (i) at least one modification within a RuvCdomain or an HNH domain, such that the engineered Bacillus smithii Cas9protein functions as a nickase; or (ii) at least one modification withina RuvC domain and an HNH domain, such that the engineered Bacillussmithii Cas9 protein is catalytically inactive.
 4. The system of claim1, wherein the engineered guide RNA comprises the nucleotide sequence ofSEQ ID NO:31.
 5. The system of claim 1, wherein the engineered Bacillussmithii Cas9 protein further comprises a chromatin modulating motif. 6.The system of claim 1, wherein the engineered Bacillus smithii Cas9protein comprises at least one modification within a RuvC domain or anHNH domain, such that the engineered Bacillus smithii Cas9 proteinfunctions as a nickase, and wherein the engineered Bacillus smithii Cas9protein further comprises a cell-penetrating domain, a marker domain, anRNA aptamer binding domain, or combination thereof.
 7. The system ofclaim 1, wherein the engineered Bacillus smithii Cas9 protein comprisesat least one modification within a RuvC domain and an HNH domain, suchthat the engineered Bacillus smithii Cas9 protein is catalyticallyinactive, and wherein the engineered Bacillus smithii Cas9 proteinfurther comprises a cell-penetrating domain, a marker domain, achromatin modulating motif, an epigenetic modification domain, atranscriptional regulation domain, an RNA aptamer binding domain, orcombination thereof.
 8. The system of claim 1, wherein the system isselected from the group consisting of: (1) a system comprising (a) anengineered Bacillus smithii Cas9 protein comprising a NLS, whichcomprises the amino acid sequence of SEQ ID NO: 2; and (b) an engineeredguide RNA, which comprises the nucleotide sequence of SEQ ID NO: 31designed to hybridize to a target sequence 5′ to a PAM comprising thesequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T; and (2) a systemcomprising (a) an engineered Bacillus smithii Cas9 protein comprising aNLS, which comprises the amino acid sequence of SEQ ID NO: 119 or SEQ IDNO: 120; and (b) an engineered guide RNA designed to hybridize to atarget sequence 5′ to a PAM comprising the sequence 5′-NNNNCAAA-3′,wherein N is A, C, G, or T.
 9. A plurality of nucleic acids encoding asystem for eukaryotic genome modification comprising (a) an engineeredBacillus smithii Cas9 protein comprising a nuclear localization signal(NLS); and (b) an engineered guide RNA, wherein the engineered guide RNAis designed to complex with the engineered Bacillus smithii Cas9 proteinand the engineered guide RNA comprises a 5′ guide sequence designed tohybridize with a target sequence in a double-stranded sequence, andwherein the target sequence is 5′ to a protospacer adjacent motif (PAM)comprising the sequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T; theplurality of nucleic acids comprising at least one nucleic acid encodingthe engineered Bacillus smithii Cas9 protein, and at least one nucleicacid encoding the engineered guide RNA.
 10. The plurality of nucleicacids of claim 9, wherein the at least one nucleic acid encoding theengineered Bacillus smithii Cas9 protein is RNA.
 11. The plurality ofnucleic acids of claim 9, wherein the at least one nucleic acid encodingthe engineered Bacillus smithii Cas9 protein is DNA.
 12. The pluralityof nucleic acids of claim 9, wherein the at least one nucleic acidencoding the engineered Bacillus smithii Cas9 protein is codon optimizedfor expression in a eukaryotic cell.
 13. The plurality of nucleic acidsof claim 12, wherein the eukaryotic cell is a human cell, a non-humanmammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, aplant cell, or a single cell eukaryotic organism.
 14. The plurality ofnucleic acids of claim 9, wherein the at least one nucleic acid encodingthe engineered guide RNA is DNA.
 15. The plurality of nucleic acids ofclaim 9, wherein the at least one nucleic acid encoding the engineeredBacillus smithii Cas9 protein is operably linked to a phage promotersequence for in vitro RNA synthesis or protein expression in a bacterialcell, and the at least one nucleic acid encoding the engineered guideRNA is operably linked to a phage promoter sequence for in vitro RNAsynthesis.
 16. The plurality of nucleic acids of claim 9, wherein the atleast one nucleic acid encoding the engineered Bacillus smithii Cas9protein is operably linked to a eukaryotic promoter sequence forexpression in a eukaryotic cell, and the at least one nucleic acidencoding the engineered guide RNA is operably linked to a eukaryoticpromoter sequence for expression in a eukaryotic cell.
 17. The pluralityof nucleic acids of claim 9, wherein the engineered guide RNA comprisesthe nucleotide sequence of SEQ ID NO:31.
 18. At least one vectorcomprising the plurality of nucleic acids of claim 9, wherein the atleast one vector is a plasmid vector, a viral vector, or aself-replicating viral RNA replicon.
 19. A eukaryotic cell comprising atleast one system comprising (a) an engineered Bacillus smithii Cas9protein comprising a nuclear localization signal (NLS); and (b) anengineered guide RNA, wherein the engineered guide RNA is designed tocomplex with the engineered Bacillus smithii Cas9 protein and theengineered guide RNA comprises a 5′ guide sequence designed to hybridizewith a target sequence in a double-stranded sequence, and wherein thetarget sequence is 5′ to a protospacer adjacent motif (PAM) comprisingthe sequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T, wherein theeukaryotic cell is a non-human mammalian cell, a plant cell, anon-mammalian vertebrate cell, an invertebrate cell, or a single celleukaryotic organism and wherein the eukaryotic cell is in vivo, ex vivo,or in vitro.
 20. A human eukaryotic cell comprising at least one systemcomprising (a) an engineered Bacillus smithii Cas9 protein comprising anuclear localization signal (NLS); and (b) an engineered guide RNA,wherein the engineered guide RNA is designed to complex with theengineered Bacillus smithii Cas9 protein and the engineered guide RNAcomprises a 5′ guide sequence designed to hybridize with a targetsequence in a double-stranded sequence, and wherein the target sequenceis 5′ to a protospacer adjacent motif (PAM) comprising the sequence5′-NNNNCAAA-3′, wherein the human eukaryotic cell is ex vivo, or invitro.
 21. A fusion protein comprising an engineered Bacillus smithiiCas9 protein linked to at least one chromatin modulating motif, whereinthe chromatin modulating motif is a high mobility group (HMG) box (HMGB)DNA binding domain, a HMG nucleosome-binding (HMGN) protein, a centralglobular domain from a histone H1 variant comprising SEQ ID NO: 74, or acombination thereof, wherein the at least one chromatin modulating motifis linked to the Cas9 protein at its N-terminus, C-terminus, or acombination thereof, wherein, together with an engineered guide RNAdesigned to complex with the Cas9 protein, the fusion protein is capableof hybridizing with a target sequence in a double-stranded sequence, andwherein the target sequence is 5′ to a protospacer adjacent motif (PAM)comprising the sequence 5′-NNNNCAAA-3′, wherein N is A, C, G, or T. 22.The fusion protein of claim 21, wherein the at least one chromatinmodulating motif is a human HMGB1 box A domain, a HMGN1 protein, a humanhistone H1 central globular domain comprising SEQ ID NO: 74, or acombination thereof.
 23. The fusion protein of claim 21, wherein the atleast one chromatin modulating motif is linked to the Cas9 proteindirectly via a chemical bond, indirectly via a linker, or a combinationthereof.
 24. The fusion protein of claim 21, further comprising at leastone nuclear localization signal, at least one cell-penetrating domain,at least one marker domain, or a combination thereof.
 25. The fusionprotein of claim 21, wherein the fusion protein comprises the amino acidsequence of SEQ ID NO: 119 or SEQ ID NO:
 120. 26. A system foreukaryotic genome modification comprising the fusion protein of claim 21and an engineered guide RNA, wherein the engineered guide RNA comprisesthe nucleotide sequence of SEQ ID NO:31.